![those whose homes lay in foreign parts, those closely connected
with the diplomatic service and the growth of the British Empire.[55]
The Chronicle was a portion of the Magazine sure of finding readers,
but there was no page more welcome to all than the brief but pithy
preface in which the editor named the chief contents, touched on
some matter of note to the readers, or urged forward the lagging
subscriber.
As the College interest widened with the ever-increasing number
of old pupils, the Chronicle became too limited a record to stand
alone. When the Magazine was about seventeen years old ‘Parerga’
appeared for the first time, telling of activities which lay outside the
immediate scope of College work, yet were due in part to the
influence of the Alma Mater, to ‘the spiritual force, the higher volition
and action.’ Miss Beale, who found in the Magazine a strong link with
her large scattered family, also in later years freely printed letters
she received from various members abroad. She did not care much
for articles on travel, writing on one occasion that she received too
many descriptions, and would like in their place to have more
records of observation in the fields of natural history and other
sciences. But she treasured letters, and showed them widely.
Indeed, it was sometimes startling for the writer of a private letter to
Miss Beale to find whole extracts published in the Magazine for all
the world to see.
Almost from the beginning there were reviews of books. These
were generally written by the editor. There were also notices of
books by old pupils. Of these Miss Beale was proud, and she never
failed to mention them, often reprinting portions of reviews by the
press; but she would not review them herself, saying, ‘Books by old
pupils claim our notice; we must leave criticism to those less
interested in the writers.’
Fortunately Miss Beale was not content with merely reviewing and
editing. Many a number of the Magazine contained a long
contribution from herself, such as an article reprinted from another
periodical, an address given at a gathering of old pupils, or at some](https://image.slidesharecdn.com/80169-250411223128-40954003/85/Informatics-In-Proteomics-1st-Edition-Sudhir-Srivastava-51-320.jpg)
![more general meeting. The first two editions of the History of the
College were also printed here. Of her articles which were not of
special College interest, the most notable were those upon
Browning. One of these, written in spring 1890, shortly after the
poet’s death, contains a brief clear statement of the value of his
philosophy. The other writers of the Magazine have been chiefly old
pupils, some of whose names, as, for example, those of Jane
Harrison, Beatrice Harraden, Bertha Synge, May Sinclair, are known
in wider fields of literature. But any who made a sincere effort were
welcomed, encouraged, and—edited. Present pupils have rarely
written, but of late an attempt has been made to secure more
contributions from these. Members of the Council, and others
connected with the College by the ties of friendship or work,
frequently helped the Magazine with papers or verses. For years
every number was enriched with a poem or article from the pen of
Mrs. James Owen, that friend whose keen intellectual interests and
strong sympathy were put so largely at Miss Beale’s service when
this literary venture was first made.
To find contributors Miss Beale went even beyond the outer circle
of the College. ‘We always hope to have some good writing in our
Magazine, thus to maintain a high standard,’ she had said at the
beginning. She liked to gain the notice of those who were eminent in
literature or science for this dearly loved literary child, and as
occasion brought her in contact with any who were distinguished for
the things she appreciated she would send them the Magazine, often
asking for a paper. Letters from people of widely differing thought
and position, acknowledging the receipt of the Magazine, are now in
the College archives. They vary in warmth and interest. The late
Bishop of Gloucester and Bristol wrote in 1889: ‘However busy I may
be, I always find time to read portions of [the Magazine], and I am
always thankful to recognise not merely the cultivated, but the wise
and—what we men specially value—the womanly tone that
characterises it. I read with much interest your article on the
Sorbonne gathering.’ Bishop Westcott in 1890 wrote, on receiving
the number containing Miss Beale’s ‘In Memoriam’ article on](https://image.slidesharecdn.com/80169-250411223128-40954003/85/Informatics-In-Proteomics-1st-Edition-Sudhir-Srivastava-52-320.jpg)
![‘These substituted verses are lovely.—Ever gratefully (1)
yrs.,
‘J. R.
‘(1) I mean, for the way you have borne with my
letters. You will not think it was because I did not like my
own work to have the other with it that I spoke as I did.’
Mr. Shorthouse also once contributed to the Magazine, sending a little
story called ‘An Apologue.’
The work entailed by the Magazine was, on the whole, pleasant and
interesting to its editor. But she was grieved sometimes if she thought
old pupils did not appreciate it, or if contributions fell short. It was not
always easy to get enough articles of the kind she desired, and the
difficulty was increased by the severe censorship she exercised. ‘About
one hour wasted in fretting over Magazine,’ runs the diary of April 2,
1891.
The Magazine was not without its faults. ‘How bad the best of us!’
says Punch, according to Ruskin. But it had the conspicuous merit of
offering encouragement to young writers, of promoting a spirit of unity,
and fostering sympathetic interest among those whose lives were
necessarily far apart. ‘We hope,’ Miss Beale had said in her first preface,
‘that the papers on work may be helpful in suggesting ways of
usefulness.’[56] This hope was practically realised. How far the young
writers profited by each other’s thoughts can be less easily gauged; but
doubtless some learned at least one lesson the Magazine was meant to
teach, that if they intended to work, they ‘must not shrink from the
hardest and most fruitful work, i.e. thinking.’[57]
Miss Beale’s influence was again extended in manifold and ever-
developing ways when, in 1883, the first meeting of former pupils was
held in the College.
At this date the number of regular pupils was five hundred. Only six
years before a proposal had been made to limit the numbers to three
hundred, but each year saw an increase, and a consequent addition to](https://image.slidesharecdn.com/80169-250411223128-40954003/85/Informatics-In-Proteomics-1st-Edition-Sudhir-Srivastava-57-320.jpg)
![high an intellectual and social standard as possible, first
amongst those of their own class. Thus reading societies,
mutual improvement societies, libraries, etc., would be
helped on by them. They would bear in mind the College
motto, “Let no man think or maintain that a man can
search too far or be too well studied in the Book of God’s
Word, or in the Book of God’s Works; but rather let men
endeavour an endless progress and proficiency in both;
only let men beware that they apply both to charity and
not to grovelling; to use and not to ostentation.”[58] Some
articles of their creed would be—(a) that influence
radiates from a centre, and hence it is a duty all through
life to continue one’s own education; (b) that the nearer
we stand in intellectual and social position, the stronger
are our ties to any, and the greater are our duties; (c)
that the worst thing one can do with any talent one
possesses is to bury it. Rules would have to be framed
concerning admission.’
Miss Beale added that secretaries to the proposed association had
already been appointed: Mrs. Ashley Smith for the general work and
organisation, Miss Flora Ker as local secretary. This announcement of
her appointment to what proved to be a very strenuous work was the
first suggestion that Mrs. Smith received that she should even undertake
it. In an article in the next Magazine Miss Beale unfolded her plan more
fully, suggesting a few rules. She proposed further that the badge of the
association should be a little brooch engraved with a figure of her
beloved Britomart.
The idea of a guild of old pupils was eagerly received, and a
committee at once formed to deal with its organisation. In all these
arrangements Miss Beale showed great strength of mind and self-control
in being able to stand aside and let others work out the details of the
scheme, even submitting her own judgment to that of the younger
ones, whom she thought called upon to do the work. Yet she was in a
true sense President of the Guild, guiding and directing where she would
not command. Indeed, this ever-growing society which multiplied
interests for her was largely her own inception, at a time when her](https://image.slidesharecdn.com/80169-250411223128-40954003/85/Informatics-In-Proteomics-1st-Edition-Sudhir-Srivastava-62-320.jpg)
![1. A scheme for educating at College a few pupils who
were worthy of education, but unable to pay the fees.
2. A scheme for taking over an elementary school in
order to work it through teachers who had been trained in
College.
3. The third scheme, which was carried, was submitted
to the Guild in these words: ‘That the corporate fund be
devoted to starting and supporting a mission in one of our
large towns, the place to be decided by the votes of the
Guild Members.’
It was but natural that President and members should have different
ideas on such an occasion. Dorothea Beale, who had never ceased to
hear and obey the call she had received as a girl to help women, and
with them the race, by means of improved education, longed to see
those she had taught and trained freely sharing with others the very
same advantages they had received. The difficulties which beset her
own youth were still fresh in her mind. The need for good teachers still
existed. She had seen the work she wanted the Guild to take up in
operation for years, knew that it did not pauperise, that it blessed giver
and receiver, and was increasingly fruitful, like good seed in good
ground. On the other hand, she had a profound suspicion of much
charitable work of the day, thinking that ‘it will quickly perish because it
does not aim at developing energy, inward power. To do for others what
they ought to do for themselves is to degrade them in the order of
creation.’[59] She could far more easily bear to see people suffering from
hunger and nakedness than from loss of will power and sense of
responsibility. This was partly, perhaps, because she did not know nor in
the least realise the miseries and difficulties of extreme poverty.
Miss Beale’s misgivings about the East End work were probably never
quite set at rest. Writing to Mrs. Charles Robinson in 1899, she said: ‘I
shall perhaps sleep two nights at St. Hilda’s East. I feel the whole
question of Settlements most difficult. It was undertaken against my
judgment, and yet the guidance all the way seems to point to its being
right. Sisters and Deaconesses are much better for this work, yet there](https://image.slidesharecdn.com/80169-250411223128-40954003/85/Informatics-In-Proteomics-1st-Edition-Sudhir-Srivastava-68-320.jpg)
![CHAPTER XI
ST. HILDA’S WORK
‘Thy kindred with the great of old.’
Tennyson, In Memoriam, lxxiv.
Those who had often the advantage of hearing Miss Beale speak,
either in general addresses to present or past pupils, or in the more
regular course of literature lessons, soon learned that there were certain
heroic names which had for her an almost romantic fascination. Among
those of great women who influenced her imagination are specially to
be remembered St. Hilda, St. Catherine of Siena, la Mère Angélique,
Mme. Guyon. Of these the most dominant, the most inspiring was that
of the great Northumbrian abbess, known to those whom she taught
and ruled by the name of ‘Mother,’ not by virtue of her office, but on
account of her signal piety and grace.[60] Hilda, the earnest student who
‘had been diligently instructed by learned men, who so loved order that
she immediately began to reduce all things to a regular system.’ Hilda,
the patron of the first English religious poet, ‘who obliged those under
her to attend much to the reading of the Holy Scriptures; who taught
the strict observance of justice and other virtues, particularly of peace
and charity.’[61] This great Hilda and her work were to Dorothea Beale
not merely romantic names, they were an ideal, an inspiration. And
when the due time came, though for the sake of Miss Newman she
hesitated for a moment over the alternative title of St. Margaret’s Hall,
the name of St. Hilda was the one she chose to grace her own
foundations. There are, possibly, members of the Ladies’ College who
felt a pang of envy when the Students’ House became St. Hilda’s
College. They could have borne to exchange the prim early Victorian
title bestowed by the godfathers of 1856 for this more inspiring name.](https://image.slidesharecdn.com/80169-250411223128-40954003/85/Informatics-In-Proteomics-1st-Edition-Sudhir-Srivastava-74-320.jpg)
![upwards and obtained his position from hard work, thus
adding to his dignity and power of teaching. And I should
follow as much as possible in these tracks.’
Eventually the ideas expressed in these letters were carried out in the
arrangement of St. Hilda’s, which became not only a home for pupils
who could not afford the normal boarding fees, but also a residence for
senior students who needed more liberty than they could have in the
other houses. By this means the house was put on a self-supporting
basis. Miss Beale could have borne with no other. The Loan Fund, up to
this time, had been the means of assisting over a hundred students.
Miss Beale now asked a few personal friends to support it, pointing out
that such a means of help was far better than any system of
scholarships, which she never ceased to dislike, and against which she
continually spoke and wrote. Her chief objections to scholarships have
been already noted.[62] She was moreover opposed to the principle of
material giving involved in the system. She only cared, at any time, to
give what would embrace and ennoble character. She thought it best
that people should pay for advantages received, thought they would
value them more, thought it made girls more careful and self-denying
when first the management of money came into their own hands, to feel
that it was not their own to do as they pleased with. A mere gift seemed
to her like a dead thing compared with the money which, lent and
returned and then lent to others, was thus used over and over again.
Yet the want of response to appeals for the Loan Fund must have been
partly due to a difference of opinion on its method rather than to want
of sympathy with Miss Beale’s aims. There are many who feel an
objection to saddling with a loan a young teacher starting on her work,
or who recognise that an unpaid loan may help to lower the standard in
money affairs, and on that account shrink from giving help in this way.
There are few indeed who could lend money so successfully as Miss
Beale could, because there are few who could so successfully command
repayment. Of the first £500 advanced by the Loan Fund, £495 was
repaid in a very few years. The pressure she would exercise for
repayment sometimes led to the wrong notion that she cared for money
for its own sake. She had at all times great skill in wringing the utmost
use out of a sum of money to promote those ends for which she lived;](https://image.slidesharecdn.com/80169-250411223128-40954003/85/Informatics-In-Proteomics-1st-Edition-Sudhir-Srivastava-78-320.jpg)
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- 9. Published in 2005 by CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2005 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 1-57444-480-8 (Hardcover) International Standard Book Number-13: 978-1-57444-480-3 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http://www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Catalog record is available from the Library of Congress Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com Taylor & Francis Group is the Academic Division of T&F Informa plc.
- 10. Dedication Dedicated to my daughters, Aditi and Jigisha, and to my lovely wife, Dr. Rashmi Gopal Srivastava
- 12. Foreword A remarkable development in the post-genome era is the re-emergence of proteomics as a new discipline with roots in old-fashioned chemistry and biochemistry, but with new branches in genomics and informatics. The appeal of proteomics stems from the fact that proteins are the most functional component encoded for in the genome and thus represent a direct path to functionality. Proteomics emphasizes the global profiling of cells, tissues, and biological fluids, but there is a long road from applying various proteomics tools to the discovery, for example, of proteins that have clinical utility as disease markers or as therapeutic targets. Given the complexity of various cell and tissue proteomes and the challenges of identifying proteins of particular interest, informatics is central to all aspects of proteomics. However, protein infor- matics is still in its early stages, as is the entire field of proteomics. Although collections of protein sequences have preceded genomic sequence data- bases by more than two decades, there is a substantial need for protein databases as basic protein information resources. There is a need for implementing algorithms, statistical methods, and computer applications that facilitate pattern recognition and biomarker discovery by integrating data from multiple sources. This book, which is dedicated to protein informatics, is intended to serve as a valuable resource for people interested in protein analysis, particularly in the context of biomedical studies. An expert group of authors has been assembled with proteomics informatics–related expertise that is highly valuable in guiding proteomic studies, particularly since currently the analysis of pro- teomics data is rather informal and largely dependent on the idiosyncrasies of the analyst. Several chapters address the need for infrastructures for proteomic research and cover the status of public protein databases and interfaces. The creation of a national virtual knowledge environment and information management systems for proteomic research is timely and clearly addressed. Issues surrounding data standardization and integration are very well presented. They are captured in a chapter that describes ongoing initiatives within the Human Proteome Organization (HUPO). A major strength of the book is in the detailed review and discussion of applications of statistical and bioinformatic tools to data analysis and data mining. Much concern at the present time surrounds the analysis of proteomics data by mass spectrometry for a variety of applications. The book shines in its presentation in several chapters of various approaches and issues surrounding mass spectrometry data analysis. Although the field of proteomics and related informatics is highly evolving, this book captures not only the current state-of-the-art but also presents a vision for where the field is heading. As a result, the contributions of the book and its com- ponent chapters will have long-lasting value. Sam Hanash, M.D. Fred Hutchinson Cancer Center Seattle, Washington
- 14. Preface The biological dictates of an organism are largely governed through the structure and function of the products of its genes, the most functional of which is the proteome. Originally defined as the analysis of the entire protein complement of a cell or tissue, proteomics now encompasses the study of expressed proteins including the identification and elucidation of their structure–function relationships under normal and disease conditions. In combination with genomics, proteomics can provide a holistic understanding of the biology underlying disease processes. Infor- mation at the level of the proteome is critical for understanding the function of specific cell types and their roles in health and disease. Bioinformatic tools are needed at all levels of proteomic analysis. The main databases serving as the targets for mass spectrometry data searches are the expressed sequence tag (EST) and the protein sequence databases, which contain protein sequence information translated from DNA sequence data. It is thought that virtually any protein that can be detected on a 2DE gel can be identified through the EST database, which contains over 2 million cDNA sequences. However, ESTs cover only a partial sequence of the protein. This poses a formidable challenge for the proteomic community and neces- sitates the need for databases with extensive coverage and search algorithms for identifying proteins/peptides with accuracy. The handling and analysis of data generated by proteomic investigations repre- sent an emerging and challenging field. New techniques and collaborations between computer scientists, biostatisticians, and biologists are called for. There is a need to develop and integrate a variety of different types of databases; to develop tools for translating raw primary data into forms suitable for public dissemination and formal data analysis; to obtain and develop user interfaces to store, retrieve, and visualize data from databases; and to develop efficient and valid methods of data analysis. The sheer volume of data to be collected and processed will challenge the usual approaches. Analyzing data of this dimension is a fairly new endeavor for statisti- cians, for which there is not an extensive technical statistical literature. There are several levels of complexity in the investigation of proteomic data, from the day-to-day interpretation of protein patterns generated by individual mea- surement systems to the query and manipulation of data from multiple experiments or information sources. Interaction with data warehouses represents another level of data interrogation. Users typically retrieve data and formulate queries to test hypoth- eses and generate conclusions. Formulating queries can be a difficult task requiring extensive syntactic and semantic knowledge. Syntactic knowledge is needed to ensure that a query is well formed and references existing relations and attributes. Semantic knowledge is needed to ensure that a query satisfies user intent. Because a user often has an incomplete understanding of the contents and structure of the data warehouse, it is necessary to provide automated techniques for query formulation that significantly reduce the amount of knowledge required by data warehouse users.
- 15. This book intends to provide a comprehensive view of informatic approaches to data storage, curation, retrieval, and mining as well as application-specific bioinformatic tools in disease detection, diagnosis, and treatment. Rapid technological advances are yielding abundant data in many formats that, because of their vast quantity and complexity, are becoming increasingly difficult to analyze. A strategic objective is to streamline the transfer of knowledge and technology to allow for data from disparate sources to be analyzed, providing new inferences about the complex role of proteomics in disease processes. Data mining, the process of knowledge extraction from data and the exploration of available data for patterns and relationships, is increasingly needed for today’s high-throughput technologies. Data architectures that support the integration of biological data files with epidemiologic profiles of human clinical responses need to be developed. The ability to develop and analyze metadata will stimulate new research theories and streamline the transfer of basic knowledge into clinical applications. It is my belief that this book will serve as a unique reference for researchers, biologists, technol- ogists, clinicians, and other health professions as it provides information on the informatics needs of proteomic research on molecular targets relevant to disease detection, diagnosis, and treatment. The nineteen chapters in this volume are contributed by eminent researchers in the field and critically address various aspects of bioinformatics and proteomic research. The first two chapters are introductory: they discuss the biological rationale for proteomic research and provide a brief overview of technologies that allow for rapid analysis of the proteome. The next five chapters describe the infrastructures that provide the foundations for proteomic research: these include the creation of a national, virtual knowledge environment and information management systems for proteomic research; the availability of public protein databases and interfaces; and the need for collaboration and interaction between academia, industry, and government agencies. Chapter 6 illustrates the power of proteomic knowledge in furthering hypoth- esis-driven cancer biomarker research through data extraction and curation. Chapter 7 and Chapter 8 provide the conceptual framework for data standardization and inte- gration and give an example of an ongoing collaborative research within the Human Proteome Organization. Chapter 9 identifies genomic and proteomic informatic tools used in deciphering functional pathways. The remaining ten chapters describe appli- cations of statistical and bioinformatic tools in data analysis, data presentation, and data mining. Chapter 10 provides an overview of a variety of proteomic data mining tools, and subsequent chapters provide specific examples of data mining approaches and their applications. Chapter 11 describes methods for quantitative analysis of a large number of proteins in a relatively large number of lung cancer samples using two-dimensional gel electrophoresis. Chapter 12 discusses the analysis of mass spec- trometric data by nonparametric inference for high-dimensional comparisons involv- ing two or more groups, based on a few samples and very few replicates from within each group. Chapter 13 discusses bioinformatic tools for the identification of proteins by searching a collection of sequences with mass spectrometric data and describes several critical steps that are necessary for the successful protein identification, which include: (a) the masses of peaks in the mass spectrum corresponding to the monoiso- topic peptide masses have to assigned; (b) a collection of sequences have to be
- 16. searched using a sensitive and selective algorithm; (c) the significance of the results have to be tested; and (d) the function of the identified proteins have to be assigned. In Chapter 14, two types of approaches are described: one based on statistical theories and another on machine learning and computational data mining tech- niques. In Chapter 15, the author discusses the problems with the currently avail- able disease classifier algorithms and puts forward approaches for scaling the data set, searching for outliers, choosing relevant features, building classification mod- els, and then determining the characteristics of the models. Chapter 16 discusses currently available computer tools that support data collection, analysis, and val- idation in a high-throughput LC-MS/MS–based proteome research environment and subsequent protein identification and quantification with minimal false-posi- tive error rates. Chapter 17 and Chapter 18 describe experimental designs, statis- tical methodologies, and computational tools for the analysis of spectral patterns in the diagnosis of ovarian and prostate cancer. Finally, Chapter 19 illustrates how quantitative analysis of fluorescence microscope images augments mainstream proteomics by providing information about the abundance, localization, move- ment, and interactions of proteins inside cells. This book has brought together a mix of scientific disciplines and specializations, and I encourage readers to expand their knowledge by reading how the combination of proteomics and bioinformatics is used to uncover interesting biology and discover clinically significant biomarkers. In a field with rapidly changing technologies, it is difficult to ever feel that one has knowledge that is current and definitive. Many chapters in this book are conceptual in nature but have been included because proteomics is an evolving science that offers much hope to researchers and patients alike. Last, but not least, I would like to acknowledge the authors for their contributions and patience. When I accepted the offer to edit this book, I was not sure we were ready for a book on proteomics as the field is continuously evolving, but the excellent contributions and enthusiasm of my colleagues have allayed my fears. The chapters in the book describe the current state-of-the-art in informatics and reflect the inter- ests, experience, and creativity of the authors. Many chapters are intimately related and therefore there may be some overlap in the material presented in each individual chapter. I would also like to acknowledge Dr. Asad Umar for his help in designing the cover for this book. Finally, I would like to express my sincere gratitude to Dr. Sam Hanash, the past president of HUPO, for his encouragement and support. Sudhir Srivastava, Ph.D., MPH, MS Bethesda, Maryland
- 18. Contributors Bao-Ling Adam Department of Microbiology and Molecular Cell Biology Eastern Virginia Medical School Norfolk, Virginia, USA Marcin Adamski Bioinformatics Program Department of Human Genetics School of Medicine University of Michigan Ann Arbor, Michigan, USA Ruedi Aebersold Institute for Systems Biology Seattle, Washington, USA R.C. Beavis Beavis Informatics Winnipeg, Manitoba, Canada David G. Beer General Thoracic Surgery University of Michigan Ann Arbor, Michigan, USA Guoan Chen General Thoracic Surgery University of Michigan Ann Arbor, Michigan, USA Chad Creighton Pathology Department University of Michigan Ann Arbor, Michigan, USA Daniel Crichton Jet Propulsion Laboratory California Institute of Technology Pasadena, California, USA Cim Edelstein Division of Public Health Services Fred Hutchinson Cancer Research Center Seattle, Washington, USA Jimmy K. Eng Division of Public Health Services Fred Hutchinson Cancer Research Center Seattle, Washington, USA J. Eriksson Department of Chemistry Swedish University of Agricultural Sciences Uppsala, Sweden Ziding Feng Division of Public Health Sciences Fred Hutchinson Cancer Research Center Seattle, Washington, USA D. Fenyö Amersham Biosciences AB Uppsala, Sweden The Rockefeller University New York, New York, USA R. Gangal SciNova Informatics Pune, Maharashtra, India
- 19. Gary L. Gilliland Biotechnology Division National Institute of Standards and Technology Gaithersburg, Maryland, USA Samir M. Hanash Division of Public Health Sciences Fred Hutchinson Cancer Research Center Seattle, Washington, USA Ben A. Hitt Correlogic Systems, Inc. Bethesda, Maryland, USA J. Steven Hughes Jet Propulsion Laboratory California Institute of Technology Pasadena, California, USA Donald Johnsey National Cancer Institute National Institutes of Health Bethesda, Maryland, USA Andrew Keller Division of Public Health Sciences Fred Hutchinson Cancer Research Center Seattle, Washington, USA Sean Kelly Jet Propulsion Laboratory California Institute of Technology Pasadena, California, USA Heather Kincaid Fred Hutchinson Cancer Research Center Seattle, Washington, USA Jeanne Kowalski Division of Oncology Biostatistics Johns Hopkins University Baltimore, Maryland, USA Peter A. Lemkin Laboratory of Experimental and Computational Biology Center for Cancer Research National Cancer Institute Frederick, Maryland, USA Xiao-jun Li Institute for Systems Biology Seattle, Washington, USA Chenwei Lin Department of Computational Biology Fred Hutchinson Cancer Research Center Seattle, Washington, USA Lance Liotta FDA-NCI Clinical Proteomics Program Laboratory of Pathology National Cancer Institute Bethesda, Maryland, USA Stephen Lockett NCI–Frederick/SAIC–Frederick Frederick, Maryland, USA Brian T. Luke SAIC-Frederick Advanced Biomedical Computing Center NCI Frederick Frederick, Maryland, USA Dale McLerran Division of Public Health Sciences Fred Hutchinson Cancer Research Center Seattle, Washington, USA Djamel Medjahed Laboratory of Molecular Technology SAIC-Frederick Inc. Frederick, Maryland, USA
- 20. Alexey I. Nesvizhskii Division of Public Health Sciences Fred Hutchinson Cancer Research Center Seattle, Washington, USA Jane Meejung Chang Oh Wayne State University Detroit, Michigan, USA Gilbert S. Omenn Departments of Internal Medicine and Human Genetics Medical School and School of Public Health University of Michigan Ann Arbor, Michigan, USA Emanuel Petricoin FDA-NCI Clinical Proteomics Program Office of Cell Therapy CBER/Food and Drug Administration Bethesda, Maryland, USA Veerasamy Ravichandran Biotechnology Division National Institute of Standards and Technology Gaithersburg, Maryland, USA John Semmes Department of Microbiology and Molecular Cell Biology Eastern Virginia Medical School Norfolk, Virginia, USA Ram D. Sriram Manufacturing Systems Integration Division National Institute of Standards and Technology Gaithersburg, Maryland, USA Sudhir Srivastava Cancer Biomarkers Research Group Division of Cancer Prevention National Cancer Institute Bethesda, Maryland, USA David J. States Bioinformatics Program Department of Human Genetics School of Medicine University of Michigan Ann Arbor, Michigan, USA Mark Thornquist Division of Public Health Sciences Fred Hutchinson Cancer Research Center Seattle, Washington, USA Mukesh Verma Cancer Biomarkers Research Group Division of Cancer Prevention National Cancer Institute Bethesda, Maryland, USA Paul D. Wagner Cancer Biomarkers Research Group Division of Cancer Prevention National Cancer Institute Bethesda, Maryland, USA Denise B. Warzel Center for Bioinformatics National Cancer Institute Rockville, Maryland, USA Nicole White Department of Pathology Johns Hopkins University Baltimore, Maryland, USA Marcy Winget Department of Population Health and Information Alberta Cancer Board Edmonton, Alberta, Canada
- 21. Yutaka Yasui Division of Public Health Sciences Fred Hutchinson Cancer Research Center Seattle, Washington, USA Mei-Fen Yeh Division of Oncology Biostatistics Johns Hopkins University Baltimore, Maryland, USA Zhen Zhang Center for Biomarker Discovery Department of Pathology Johns Hopkins University Baltimore, Maryland, USA
- 22. Contents Chapter 1 The Promise of Proteomics: Biology, Applications, and Challenges.......................1 Paul D. Wagner and Sudhir Srivastava Chapter 2 Proteomics Technologies and Bioinformatics.........................................................17 Sudhir Srivastava and Mukesh Verma Chapter 3 Creating a National Virtual Knowledge Environment for Proteomics and Information Management........................................................31 Daniel Crichton, Heather Kincaid, Sean Kelly, Sudhir Srivastava, J. Steven Hughes, and Donald Johnsey Chapter 4 Public Protein Databases and Interfaces.................................................................53 Jane Meejung Chang Oh Chapter 5 Proteomics Knowledge Databases: Facilitating Collaboration and Interaction between Academia, Industry, and Federal Agencies............................79 Denise B. Warzel, Marcy Winget, Cim Edelstein, Chenwei Lin, and Mark Thornquist Chapter 6 Proteome Knowledge Bases in the Context of Cancer ........................................109 Djamel Medjahed and Peter A. Lemkin Chapter 7 Data Standards in Proteomics: Promises and Challenges ....................................141 Veerasamy Ravichandran, Ram D. Sriram, Gary L. Gilliland, and Sudhir Srivastava Chapter 8 Data Standardization and Integration in Collaborative Proteomics Studies ........163 Marcin Adamski, David J. States, and Gilbert S. Omenn
- 23. Chapter 9 Informatics Tools for Functional Pathway Analysis Using Genomics and Proteomics.....................................................................................193 Chad Creighton and Samir M. Hanash Chapter 10 Data Mining in Proteomics ...................................................................................205 R. Gangal Chapter 11 Protein Expression Analysis..................................................................................227 Guoan Chen and David G. Beer Chapter 12 Nonparametric, Distance-Based, Supervised Protein Array Analysis .......................................................................................................255 Mei-Fen Yeh, Jeanne Kowalski, Nicole White, and Zhen Zhang Chapter 13 Protein Identification by Searching Collections of Sequences with Mass Spectrometric Data ..............................................................................267 D. Fenyö, J. Eriksson, and R.C. Beavis Chapter 14 Bioinformatics Tools for Differential Analysis of Proteomic Expression Profiling Data from Clinical Samples................................................277 Zhen Zhang Chapter 15 Sample Characterization Using Large Data Sets..................................................293 Brian T. Luke Chapter 16 Computational Tools for Tandem Mass Spectrometry–Based High-Throughput Quantitative Proteomics ...........................................................335 Jimmy K. Eng, Andrew Keller, Xiao-jun Li, Alexey I. Nesvizhskii, and Ruedi Aebersold Chapter 17 Pattern Recognition Algorithms and Disease Biomarkers....................................353 Ben A. Hitt, Emanuel Petricoin, and Lance Liotta
- 24. Chapter 18 Statistical Design and Analytical Strategies for Discovery of Disease-Specific Protein Patterns..........................................................................367 Ziding Feng, Yutaka Yasui, Dale McLerran, Bao-Ling Adam, and John Semmes Chapter 19 Image Analysis in Proteomics...............................................................................391 Stephen Lockett Index......................................................................................................................433
- 26. 1 1 The Promise of Proteomics: Biology, Applications, and Challenges Paul D. Wagner and Sudhir Srivastava CONTENTS 1.1 Introduction ......................................................................................................1 1.2 Why Is Proteomics Useful?.............................................................................2 1.3 Gene–Environment Interactions.......................................................................3 1.4 Organelle-Based Proteomics............................................................................4 1.5 Cancer Detection..............................................................................................5 1.6 Why Proteomics Has Not Succeeded in the Past: Cancer as an Example......................................................................................6 1.7 How Have Proteomic Approaches Changed over the Years? .........................7 1.8 Future of Proteomics in Drug Discovery, Screening, Early Detection, and Prevention..............................................................................11 References................................................................................................................13 1.1 INTRODUCTION In the 19th century, the light microscope opened a new frontier in the study of diseases, allowing scientists to look deep into the cell. The science of pathology (the branch of medicine that deals with the essential nature of disease) expanded to include the study of structural and functional changes in cells, and diseases could be attributed to recognizable changes in the cells of the body. At the start of the 21st century, the molecular-based methods of genomics and proteomics are bringing about a new revolution in medicine. Diseases will be described in terms of patterns of abnormal genetic and protein expression in cells and how these cellular alterations affect the molecular composition of the surrounding environment. This new pathol- ogy will have a profound impact on the practice of medicine, enabling physicians to determine who is at risk for a specific disease, to recognize diseases before they have invaded tissues, to intervene with agents or treatments that may prevent or
- 27. 2 Informatics in Proteomics delay disease progression, to guide the choice of therapies, and to assess how well a treatment is working. Cancer is one of the many diseases whose treatment will be affected by these molecular approaches. Currently available methods can only detect cancers that have achieved a certain size threshold, and in many cases, the tumors, however small, have already invaded blood vessels or spread to other parts of the body. Molecular markers have the potential to find tumors in their earliest stages of development, even before the cell’s physical appearance has changed. Molecular-based detection methods will also change our definition of cancer. For example, precancerous changes in the uterine cervix are called such because of specific architectural and cytological changes. In the future, we may be able to define the expression patterns of specific cellular proteins induced by human papillomavirus that indicate the cells are beginning to progress to cancer. We may also be able to find molecular changes that affect all the tissues of an organ, putting the organ at risk for cancer. In addition to improving the physician’s ability to detect cancers early, molecular technologies will help doctors determine which neoplastic lesions are most likely to progress and which are not destined to do so — a dilemma that confronts urologists in the treatment of prostate cancer. Accurate discrimination will help eliminate overtreatment of harmless lesions. By revealing the metastatic potential of tumors and their corresponding preneoplastic lesions, molecular-based methods will fill a knowledge gap impossible to close with traditional histopathology. If these advances are made and new screening tests are developed, then one day we may be able to identify and eliminate the invasive forms of most malignant epithelial tumors. 1.2 WHY IS PROTEOMICS USEFUL? Mammalian systems are much more complex than can be deciphered by their genes alone, and the biological dictates of an organism are largely governed through the function of proteins. In combination with genomics, proteomics can provide a holistic understanding of the biology of cells, organisms, and disease processes. The term “proteome” came into use in the mid 1990s and is defined as the protein complement of the genome. Although proteomics was originally used to describe methods for large-scale, high-throughput protein separation and identification,1 today proteomics encompasses almost any method used to characterize proteins and deter- mine their functions. Information at the level of the proteome is critical for under- standing the function of specific cell types and their roles in health and disease. This is because proteins are often expressed at levels and forms that cannot be predicted from mRNA analysis. Proteomics also provides an avenue to understand the inter- action between a cell’s functional pathways and its environmental milieu, indepen- dent of any changes at the RNA level. It is now generally recognized that expression analysis directly at the protein level is necessary to unravel the critical changes that occur as part of disease pathogenesis. Currently there is much interest in the use of molecular markers or biomarkers for disease diagnosis and prognosis. Biomarkers are cellular, biochemical, and molecular alterations by which normal, abnormal, or simply biologic processes can be recognized or monitored. These alterations should be able to objectively measure
- 28. The Promise of Proteomics: Biology, Applications, and Challenges 3 and evaluate normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. Proteomics is valuable in the discovery of biomarkers as the proteome reflects both the intrinsic genetic program of the cell and the impact of its immediate environment. Protein expression and function are subject to modulation through transcription as well as through translational and posttranslational events. More than one messenger RNA can result from one gene through differential splicing, and proteins can undergo more than 200 types of posttranslation modifications that can affect function, protein–protein and protein– ligand interactions, stability, targeting, or half-life.2 During the transformation of a normal cell into a neoplastic cell, distinct changes occur at the protein level that range from altered expression, differential modification, changes in specific activity, and aberrant localization, all of which affect cellular function. Identifying and understanding these changes is the underlying theme in cancer proteomics. The deliverables include identification of biomarkers that have utility both for early detection and for determining therapy. While proteomics has traditionally dealt with quantitative analysis of protein expression, more recently proteomics has been viewed to encompass structural analyses of proteins.3 Quantitative proteomics strives to investigate the changes in protein expression in different physiological states such as in healthy and diseased tissue or at different stages of the disease. This enables the identification of state- and stage-specific proteins. Structural proteomics attempts to uncover the structure of proteins and to unravel and map protein–protein interactions. Proteomics provides a window to pathophysiological states of cells and their microenvironments and reflects changes that occur as disease-causing agents interact with the host environ- ment. Some examples of proteomics are described below. 1.3 GENE–ENVIRONMENT INTERACTIONS Infectious diseases result from interactions between the host and pathogen, and understanding these diseases requires understanding not only alterations in gene and protein expressions within the infected cells but also alterations in the sur- rounding cells and tissues. Although genome and transcriptome analyses can pro- vide a wealth of information on global alterations in gene expression that occur during infections, proteomic approaches allow the monitoring of changes in protein levels and modifications that play important roles in pathogen–host interactions. During acute stages of infection, pathogen-coded proteins play a significant role, whereas in the chronic infection, host proteins play the dominating role. Viruses, such as hepatitis B (HBV), hepatitis C (HCV), and human papillomavirus (HPV), are suitable for proteomic analysis because they express only eight to ten major genes.4,5 Analyzing a smaller number of genes is easier than analyzing the proteome of an organism with thousands of genes.6–8 For example, herpes simplex virus type 1 (HSV-1) infection induces severe alterations of the translational apparatus, includ- ing phosphorylation of ribosomal proteins and the association of several nonribo- somal proteins with the ribosomes.9–12 Whether ribosomes themselves could con- tribute to the HSV-1–induced translational control of host and viral gene expression has been investigated. As a prerequisite to test this hypothesis, the investigators
- 29. 4 Informatics in Proteomics undertook the identification of nonribosomal proteins associated with the ribosomes during the course of HSV-1 infection. Two HSV-1 proteins, VP19C and VP26, that are associated to ribosomes with different kinetics were identified. Another nonri- bosomal protein identified was the poly(A)-binding protein 1 (PAB1P). Newly synthesized PAB1P continued to associate to ribosomes throughout the course of infection. This finding attests to the need for proteomic information for structural and functional characterization. Approximately 15% of human cancers (about 1.5 million cases per year, world- wide) are linked to viral, bacterial, or other pathogenic infections.13 For cancer development, infectious agents interact with host genes and sets of infectious agent-specific or host-specific genes are expressed. Oncogenic infections increase the risk of cancer through expression of their genes in the infected cells. Occasion- ally, these gene products have paracrine effects, leading to neoplasia in neighboring cells. More typically, it is the infected cells that become neoplastic. These viral, bacterial, and parasitic genes and their products are obvious candidates for pharma- cologic interruptions or immunologic mimicry, promising approaches for drugs and vaccines. By understanding the pathways involved in the infectious agent–host interaction leading to cancer, it would be possible to identify targets for intervention. 1.4 ORGANELLE-BASED PROTEOMICS Eukaryotic cells contain a number of organelles, including nucleoli, mitochondria, smooth and rough endoplasmic reticula, Golgi apparatus, peroxisomes, and lysosomes. The mitochondria are among the largest organelles in the cell. Mitochondrial dys- function has been frequently reported in cancer, neurodegenerative diseases, diabetes, and aging syndromes.14–16 The mitochondrion genome (16.5 Kb) codes only for a small fraction (estimated to be 1%) of the proteins housed within this organelle. The other proteins are encoded by the nuclear DNA (nDNA) and transported into the mitochondria. Thus, a proteomic approach is needed to fully understand the nature and extent of mutated and modified proteins found in the mitochondria of diseased cells. According to a recent estimate, there are 1000 to 1500 polypeptides in the human mitochondria.17–20 This estimate is based on several lines of evidence, including the existence of at least 800 distinct proteins in yeast and Arabidopsis thaliana mitochondria18,19 and the identification of 591 abundant mouse mitochondrial proteins.20 Investigators face a number of challenges in organelle proteome characterization and data analysis. A complete characterization of the posttranslational modifications that mitochondrial proteins undergo is an enormous and important task, as all of these modifications cannot be identified by a single approach. Differences in post- translational modifications are likely to be associated with the onset and progression of various diseases. In addition, the mitochondrial proteome, although relatively simple, is made up of complex proteins located in submitochondrial compartments. Researchers will need to reduce the complexity to subproteomes by fractionation and analysis of various compartments. A number of approaches are focusing on specific components of the mitochondria, such as isolation of membrane proteins, affinity labeling, and isolation of redox proteins,21 or isolation of large complexes.22
- 30. The Promise of Proteomics: Biology, Applications, and Challenges 5 Other approaches may combine expression data from other species, such as yeast, to identify and characterize the human mitochondrial proteome.23,24 The need to identify mitochondrial proteins associated with or altered during the development and progression of cancer is compelling. For example, mitochondrial dysfunction has been frequently associated with transport of proteins, such as cyto- chrome c. Mitochondrial outer membrane permeabilization by pro-apoptotic proteins, such as Bax or Bak, results in the release of cytochrome c and the induction of apoptosis. An altered ratio of anti-apoptotic proteins (e.g., Bcl-2) to pro-apoptotic proteins (e.g., Bax and Bak) promotes cell survival and confers resistance to therapy.25 1.5 CANCER DETECTION Molecular markers or biomarkers are currently used for cancer detection, diagnosis, and monitoring therapy and are likely to play larger roles in the future. In cancer research, a biomarker refers to a substance or process that is indicative of the presence of cancer in the body. It might be a molecule secreted by the malignancy itself, or it can be a specific response of the body to the presence of cancer. The biological basis for usefulness of biomarkers is that alterations in gene sequence or expression and in protein expression and function are associated with every type of cancer and with its progression through the various stages of development. Genetic mutations, changes in DNA methylation, alterations in gene expression, and alterations in protein expression or modification can be used to detect cancer, determine prognosis, and monitor disease progression and therapeutic response. Currently, DNA-based, RNA-based, and protein-based biomarkers are used in cancer risk assessment and detection. The type of biomarker used depends both on the application (i.e., risk assessment, early detection, prognosis, or response to therapy) and the availability of appropriate biomarkers. The relative advantages and disad- vantages of genomic and proteomic approaches have been widely discussed, but since a cell’s ultimate phenotype depends on the functions of expressed proteins, proteomics has the ability to provide precise information on a cell’s phenotype. Tumor protein biomarkers are produced either by the tumor cells themselves or by the surrounding tissues in response to the cancer cells. More than 80% of human tumors (colon, lung, prostate, oral cavity, esophagus, stomach, uterine, cervix, and bladder) originate from epithelial cells, often at the mucosal surface. Cells in these tumors secrete proteins or spontaneously slough off into blood, sputum, or urine. Secreted proteins include growth factors, angiogenic proteins, and proteases. Free DNA is also released by both normal and tumor cells into the blood and patients with cancer have elevated levels of circulating DNA. Thus, body fluids such as blood and urine are good sources for cancer biomarkers. That these fluids can be obtained using minimally invasive methods is a great advantage if the biomarker is to be used for screening and early detection. From a practical point of view, assays of protein tumor biomarkers, due to their ease of use and robustness, lend themselves to routine clinical practice, and histor- ically tumor markers have been proteins. Indeed, most serum biomarkers used today are antibody-based tests for epithelial cell proteins. Two of the earliest and most widely used cancer biomarkers are PSA and CA25. Prostate-specific antigen (PSA)
- 31. 6 Informatics in Proteomics is a secreted protein produced by epithelial cells within the prostate. In the early 1980s it was found that sera from prostate cancer patients contain higher levels of PSA than do the sera of healthy individuals. Since the late 1980s, PSA has been used to screen asymptomatic men for prostate cancer and there has been a decrease in mortality rates due to prostate cancer. How much of this decrease is attributable to screening with PSA and how much is due to other factors, such as better therapies, is uncertain. Although PSA is the best available serum biomarker for prostate cancer and the only one approved by the FDA for screening asymptomatic men, it is far from ideal. Not all men with prostate cancer have elevated levels of PSA; 20 to 30% of men with prostate cancer have normal PSA levels and are misdiagnosed. Con- versely, because PSA levels are increased in other conditions, such as benign pros- tatic hypertrophy and prostatitis, a significant fraction of men with elevated levels of PSA do not have cancer and undergo needless biopsies. The CA125 antigen was first detected over 20 years ago; CA125 is a mucin-like glycoprotein present on the cell surface of ovarian tumor cells that is released into the blood.26 Serum CA125 levels are elevated in about 80% of womenwith epithelial ovarian cancer but in less than 1% of healthy women. However, the CA125 test only returns a positive result for about 50% of Stage I ovarian cancer patients and is, therefore, not useful by itself as an early detection test.27 Also, CA125 is elevated in a number of benign conditions, which diminishes its usefulness in the initial diagnosis of ovarian cancer. Despite these limitations, CA125 is considered to be one of the best available cancer serum markers and is used primarily in the man- agement of ovarian cancer. Falling CA125 following chemotherapy indicates that the cancer is responding to treatment.28 Other serum protein biomarkers, such as alpha fetoprotein (AFP) for hepatocellular carcinoma and CA15.3 for breast cancer, are also of limited usefulness as they are elevated in some individuals without cancer, and not all cancer patients have elevated levels. 1.6 WHY PROTEOMICS HAS NOT SUCCEEDED IN THE PAST: CANCER AS AN EXAMPLE The inability of these protein biomarkers to detect all cancers (false negatives) reflects both the progressive nature of cancer and its heterogeneity. Cancer is not a single disease but rather an accumulation of several events, genetic and epigenetic, arising in a single cell over a long period of time. Proteins overexpressed in late stage cancers may not be overexpressed in earlier stages and, therefore, are not useful for early cancer detection. For example, the CA125 antigen is not highly expressed in many Stage I ovarian cancers. Also, because tumors are heterogeneous, the same sets of proteins are not necessarily overexpressed in each individual tumor. For example, while most patients with high-grade prostate cancers have increased levels of PSA, approximately 15% of these patients do not have an elevated PSA level. The reciprocal problem of biomarkers indicating the presence of cancer when none is present (false positives) results because these proteins are not uniquely produced by tumors. For example, PSA is produced by prostatitis (inflammation of the prostate) and benign prostatic hyperplasia (BPH), and elevated CA125 levels are caused by endometriosis and pelvic inflammation.
- 32. The Promise of Proteomics: Biology, Applications, and Challenges 7 The performance of any biomarker can be described in terms of its specificity and sensitivity. In the context of cancer biomarkers, sensitivity refers to the proportion of case subjects (individuals with confirmed disease) who test positive for the biom- arker, and specificity refers to the proportion of control subjects (individuals without disease) who test negative for the biomarker. An ideal biomarker test would have 100% sensitivity and specificity; i.e., everyone with cancer would have a positive test, and everyone without cancer would have a negative test. None of the currently available protein biomarkers achieve 100% sensitivity and specificity. For example, as described above, PSA tests achieve 70 to 90% sensitivity and only about 25% specificity, which results in many men having biopsies when they do not have detectable prostrate cancer. The serum protein biomarker for breast cancer CA15.3 has only 23% sensitivity and 69% specificity. Other frequently used terms are positive predictive value (PPV), the chance that a person with a positive test has cancer, and negative predictive value (NPV), the chance that a person with a negative test does not have cancer. PPV is affected by the prevalence of disease in the screened popu- lation. For a given sensitivity and specificity, the higher the prevalence, the higher the PPV. Even when a biomarker provides high specificity and sensitivity, it may not be useful for screening the general population if the cancer has low prevalence. For example, a biomarker with 100% sensitivity and 95% specificity has a PPV of only 17% for a cancer with 1% prevalence (only 17 out of 100 people with a positive test for the biomarker actually have cancer) and 2% for a cancer with 0.1% prevalence. The prevalence of ovarian cancer in the general population is about 0.04%. Thus, a biomarker used to screen the general population must have significantly higher spec- ificity and sensitivity than a biomarker used to monitor an at-risk population. 1.7 HOW HAVE PROTEOMIC APPROACHES CHANGED OVER THE YEARS? Currently investigators are pursuing three different approaches to develop biomarkers with increased sensitivity and specificity. The first is to improve on a currently used biomarker. For instance, specificity and sensitivity of PSA may be improved by measurement of its complex with alpha(1)-antichymotrypsin; patients with benign prostate conditions have more free PSA than bound, while patients with cancer have more bound PSA than free.29 This difference is thought to result from differences in the type of PSA released intothe circulation by benign and malignant prostatic cells. Researchers are also trying to improve the specificity and sensitivity of PSA by incorporating age- and race-specific cut points and by adjusting serum PSA concen- tration by prostatic volume (PSA density). The second approach is to discover and validate new biomarkers that have improved sensitivity and specificity. Many inves- tigators are actively pursuing new biomarkers using a variety of new and old tech- nologies. The third approach is to use a panel of biomarkers, either by combining several individually identified biomarkers or by using mass spectrometry to identify a pattern of protein peaks in sera that can be used to predict the presence of cancer or other diseases. High-throughput proteomic methodologies have the potential to revolutionize protein biomarker discovery and to allow for multiple markers to be assayed simultaneously.
- 33. 8 Informatics in Proteomics In the past, researchers have mostly used a one-at-time approach to biomarker discovery. They have looked for differences in the levels of individual proteins in tissues or blood from patients with disease and from healthy individuals. The choice of proteins to examine was frequently based on biological knowledge of the cancer and its interaction with surrounding tissues. This approach is laborious and time consuming, and most of the biomarkers discovered thus far do not have sufficient sensitivity and specificity to be useful for early cancer detection. A mainstay of protein biomarker discovery has been two-dimensional gel electrophoresis (2DE). The traditional 2DE method is to separately run extracts from control and diseased tissues or cells and to compare the relative intensities of the various protein spots on the stained gels. Proteins whose intensities are significantly increased or decreased in diseased tissues are identified using mass spectrometry. For example, 2DE was recently used to identify proteins that are specifically overexpressed in colon cancer.30 The limitations of the 2DE approach are well known: the gels are difficult to run reproducibly, a significant fraction of the proteins either do not enter the gels or are not resolved, low-abundance proteins are not detected, and relatively large amounts of sample are needed. A number of modifications have been made to overcome these limitations, including fractionation of samples prior to 2DE, the use of immobilized pH gradients, and labeling proteins from control and disease cells with different fluorescent dyes and then separating them on the same gel (differential in-gel elec- trophoresis; DIGE). An additional difficulty is contamination from neighboring stromal cells that can confound the detection of tumor-specific markers. Laser capture microdissection (LCD) can be used to improve the specificity of 2DE, as it allows for the isolation of pure cell populations; however, it further reduces the amount of sample available for analysis. Even with these modifications, 2DE is a relatively low throughput methodology that only samples a subset of the proteome, and its applicability for screening and diagnosis is very limited. A number of newer methods for large-scale protein analysis are being used or are under development. Several of these rely on mass spectrometry and database interrogation. Mass spectrometers work by imparting an electrical charge to the analytes (e.g., proteins or peptides) and then sending the charged particles though a mass analyzer. A time of flight (TOF) mass spectrometer measures the time it takes a charged particle (protein or peptide) to reach the detector; the higher the mass the longer the flight time. A mixture of proteins or peptides analyzed by TOF generates a spectrum of protein peaks. TOF mass spectrometers are used to analyze peptide peaks generated by protease digestion of proteins resolved on 2DE. A major advance in this methodology is matrix-assisted laser desorption ionization (a form of soft ionization), which allows for the ionization of larger biomolecules such as proteins and peptides. TOF mass spectrometers are also used to identify peptides eluted from HPLC columns. With tandem mass spectrometers (MS/MS), a mixture of charged peptides is separated in the first MS according to their mass-to-charge ratios, generating a list of peaks. In the second MS, the spectrometer is adjusted so that a single mass-to-charge species is directed to a collision cell to generate fragment ions, which are then separated by their mass-to-charge ratios. These patterns are compared to databases to identify the peptide and its parent protein. Liquid chromatography
- 34. The Promise of Proteomics: Biology, Applications, and Challenges 9 combined with MS or MS/MS (LC-MS and LC-MS/MS) is currently being used as an alternative to 2DE to analyze complex protein mixtures. In this approach, a mixture of proteins is digested with a protease, and the resulting peptides are then fractionated by liquid chromatography (typically reverse-phase HPLC) and analyzed by MS/MS and database interrogation. A major limitation to this approach is the vast number of peptides generated when the initial samples contain a large number of proteins. Even the most advanced LC-MS/MS systems cannot resolve and analyze these complex peptide mixtures, and currently it is necessary to either prefractionate the proteins prior to proteolysis or to enrich for certain types of peptides (e.g., phosphorylated, glycoslylated, or cysteine containing) prior to liquid chromatography. Although the use of mass spectrometry has accelerated the pace of protein identification, it is not inherently quantitative and the amounts of peptides ionized vary. Thus, the signal obtained in the mass spectrometer cannot be used to measure the amount of protein in the sample. Several comparative mass spectrometry methods have been developed to determine the relative amounts of a particular peptide or protein in two different samples. These approaches rely on labeling proteins in one sample with a reagent containing one stable isotope and labeling the proteins in the other sample with the same reagent containing a different stable isotope. The samples are then mixed, processed, and analyzed together by mass spectrometry. The mass of a peptide from one sample will be different by a fixed amount from the same peptide from the other sample. One such method (isotope-coded affinity tags; ICAT) modifies cysteine residues with an affinity reagent that contains either eight hydrogen or eight deuterium atoms.31 Other methods include digestion in 16O and 18O water and culturing cells in 12C- and 13C-labeled amino acids. Although the techniques described thus far are useful for determining proteins that are differently expressed in control and disease, they are expensive, relatively low throughput, and not suitable for routine clinical use. Surface-enhanced laser description ionization time-of-flight (SELDI-TOF) and protein chips are two pro- teomic approaches that have the potential to be high throughput and adaptable to clinical use. In the SELDI-TOF mass spectrometry approach, protein fractions or body fluids are spotted onto chromatographic surfaces (ion exchange, reverse phase, or metal affinity) that selectively bind a subset of the proteins (Ciphergen® Protein- Chip Arrays). After washing to remove unbound proteins, the bound proteins are ionized and analyzed by TOF mass spectrometry. This method has been used to identify disease-related biomarkers, including the alpha chain of haptoglobin (Hp-alpha) for ovarian cancer32 and alpha defensin for bladder cancer. Other inves- tigators are using SELDI-TOF to acquire proteomic patterns from whole sera, urine, or other body fluids. The complex patterns of proteins obtained by the TOF mass spectrometer are analyzed using pattern recognition algorithms to identify a set of protein peaks that can be used to distinguish disease from control. With this approach, protein identification and characterization are not necessary for development of clin- ical assays, and a SELDI protein profile may be sufficient for screening. For example, this method has been reported to identify patients with Stage I ovarian cancer with 100% sensitivity and 95% specificity.27 Similar, albeit less dramatic, results have been reported for other types of cancer.28,33–36 At this time, it is uncertain whether SELDI protein profiling will prove to be as valuable a diagnostic tool as the initial
- 35. 10 Informatics in Proteomics reports have suggested. A major technical issue is the reproducibility of the protein profiles. Variability between SELDI-TOF instruments, in the extent of peptide ion- ization, in the chips used to immobilize the proteins, and in sample processing, can contribute to the lack of reproducibility. There is concern that the protein peaks identified by SELDI and used for discriminating between cancer and control are not derived from the tumor per se but rather from the body’s response to the cancer (epiphenomena) and that they may not be specific for cancer; inflammatory condi- tions and benign pathologies may elicit the same bodily responses.37,38 Most known tumor marker proteins in the blood are on the order of ng/ml (PSA above 4 ng/ml and alpha fetoprotein above 20 ng/ml are considered indicators of, respectively, prostate and hepatocellular cancers). The SELDI-TOF peptide peaks typically used to distinguish cancer from control are relatively large peaks representing proteins present in the serum on the order of μg to mg/ml; these protein peaks may result from cancer-induced proteolysis or posttranslational modification of proteins nor- mally present in sera. Although identification of these discriminating proteins may not be necessary for this “black-box” approach to yield a clinically useful diagnostic test, identifying these proteins may help elucidate the underlying pathology and lead to improved diagnostic tests. Potential advantages of the SELDI for clinical assays are that it is high throughput, it is relatively inexpensive, and it uses minimally invasive specimens (blood, urine, sputum). Interest in protein chips in part reflects the success of DNA microarrays. While these two methodologies have similarities, a number of technical and biological differences exist that make the practical application of protein chips or arrays chal- lenging. Proteins, unlike DNA, must be captured in their native conformation and are easily denatured irreversibly. There is no method to amplify their concentrations, and their interactions with other proteins and ligands are less specific and of variable affinity. Current bottlenecks in creating protein arrays include the production (expres- sion and purification) of the huge diversity of proteins that will form the array elements, methods to immobilize proteins in their native states on the surface, and lack of detection methods with sufficient sensitivity and accuracy. To date, the most widely used application of protein chips are antibody microarrays that have the potential for high-throughput profiling of a fixed number of proteins. A number of purified, well-characterized antibodies are spotted onto a surface and then cell extracts or sera are passed over the surface to allow for the antigen to bind to the specific, immobilized antibodies. The bound proteins are detected either by using secondary antibodies against each antigen or by using lysates that are tagged with fluorescent or radioactive labels. A variation that allows for direct comparison between two different samples is to label each extract with a different fluorescent dye, which is then mixed prior to exposure to the antibody array. A significant problem with antibody arrays is lack of specificity; the immobilized antibodies cross react with proteins other than the intended target. The allure of protein chips is their potential to rapidly analyze multiple protein markers simultaneously at a moderate cost. As discussed earlier, most currently available cancer biomarkers lack sufficient sensitivity and specificity for use in early detection, especially to screen asymptom- atic populations. One approach to improve sensitivity and specificity is to use a panel of biomarkers. It is easy to envision how combining biomarkers can increase
- 36. The Promise of Proteomics: Biology, Applications, and Challenges 11 sensitivity if they detect different pathological processes or different stages of cancer, and one factor to consider in developing such a panel is whether the markers are complementary. However, simply combining two biomarkers will more than likely decrease specificity and increase the number of false positives. Reducing their cutoff values (the concentration of a biomarker that is used as an indication of the presence of cancer) can be useful to reduce the number of false positives. A useful test for evaluating a single biomarker or panel of biomarkers is the receiver operating characteristic (ROC) curve. An ROC curve is a graphical display of false-positive rates and true-positive rates from multiple classification rules (different cutoff values for the various biomarkers). Each point on the graph corresponds to a different classification rule. In addition to analyzing individually measured markers, ROC curves can be used to analyze SELDI-TOF proteomic profiles.39 The measurement and analysis of biomarker panels will be greatly facilitated by high-throughput technologies such as protein arrays, microbeads with multiple antibodies bound to them, and mass spectrometry. It is in these areas that a number of companies are concentrating their efforts, as not only must a biomarker or panel of biomarkers have good specificity and sensitivity, there must be an efficient and cost-effective method to assay them. 1.8 FUTURE OF PROTEOMICS IN DRUG DISCOVERY, SCREENING, EARLY DETECTION, AND PREVENTION Proteomics has benefited greatly from the development of high-throughput meth- ods to simultaneously study thousands of proteins. The successful application of proteomics to medical diagnostics will require the combined efforts of basic researchers, physicians, pathologists, technology developers, and information sci- entists (Figure 1.1). However, its application in clinics will require development FIGURE 1.1 Application of medical proteomics: Interplay between various disciplines and expertise is the key to developing tools for detection, diagnosis, and treatment of cancer. Technologist Information Scientist Basic Scientist Physician/Scientist Cancer Biorespository BIOMARKERS DIAGNOSTICS THERAPEUTICS
- 37. 12 Informatics in Proteomics of test kits based on pattern analysis, single molecule detection, or multiplexing of several clinical acceptable tests, such as ELISA, for various targets in a sys- tematic way under rigorous quality control regimens (Figure 1.2). Interperson heterogeneity is a major hurdle when attempting to discover a disease-related biomarker within biofluids such as serum. However, the coupling of high-through- put technologies with protein science now enables samples from hundreds of patients to be rapidly compared. Admittedly, proteomic approaches cannot remove the “finding a needle in a haystack” requirement for discovering novel biomarkers; however, we now possess the capability to inventory components within the “haystack” at an unprecedented rate. Indeed, such capabilities have already begun to bear fruits as our knowledge of the different types of proteins within serum is growing exponentially and novel technologies for diagnosing cancers using pro- teomic technologies are emerging. Is the development of methods capable of identifying thousands of proteins in a high-throughput manner going to lead to novel biomarkers for the diagnosis of early stage diseases or is the amount of data that is accumulated in such studies going to be overwhelming? The answer to this will depend on our ability to develop and successfully deploy bioinformatic tools. Based on the rate at which interesting leads are being discovered, it is likely that not only will biomarkers with better sensitivity and specificity be identified but individuals will be treated using custom- ized therapies based on their specific protein profile. The promise of proteomics for discovery is its potential to elucidate fundamental information on the biology of cells, signaling pathways, and disease processes; to identify disease biomarkers and new drug targets; and to profile drug leads for efficacy and safety. The promise of FIGURE 1.2 Strategies in medical proteomics: Steps in identification of detection targets and the development of clinical assays. Protein Profiling Define Protein Changes 1. 2DE 2. SELDI-TOF-MS 3. LC-coupled MS Bio-informatics Bio-computation Databases Protein Identification 1. Nano-LC-coupled SELDI-MS 2. CapLC-MS/MS 3. TOF-MS Assay Development 1. ELISA 2. SELDI-based 3. Ab arrays Functional Analysis 1. Protein-protein interaction 2. Cellular targeting 3. Protein-ligand interactions
- 38. The Promise of Proteomics: Biology, Applications, and Challenges 13 proteomics for clinical use is the refinement and development of protein-based assays that are accurate, sensitive, robust, and high throughput. Since many of the proteomic technologies and data management tools are still in their infancy, their validations and refinements are going to be the most important tasks in the future. REFERENCES 1. Wasinger, V.C., Cordwell, S.J., Cerpa-Poljak, A., et al. Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis, 16, 1090–1094, 1995. 2. Banks, R.E., Dunn, M.J., Hochstrasser, D.F., et al. Proteomics: New perspectives, new biomedical opportunities. Lancet, 356, 1749–1756, 2000. 3. Anderson, N.L., Matheson, A.D., and Steiner, S. Proteomics: Applications in basic and applied biology. Curr. Opin. Biotechnol., 11, 408–412, 2000. 4. Genther, S.M., Sterling, S., Duensing, S., Munger, K., Sattler, C., and Lambert, P.F. Quantitative role of the human papillomavirus type 16 E5 gene during the productive stage of the viral life cycle. J. Virol., 77, 2832–2842, 2003. 5. Middleton, K., Peh, W., Southern, S., et al. Organization of human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers. J. Virol., 77, 10186–10201, 2003. 6. Verma, M., Lambert, P.F., and Srivastava, S.K. Meeting highlights: National Cancer Institute workshop on molecular signatures of infectious agents. Dis. Markers, 17, 191–201, 2001. 7. Verma, M. and Srivastava, S. New cancer biomarkers deriving from NCI early detec- tion research. Recent Results Canc. Res., 163, 72–84; discussion, 264–266, 2003. 8. Verma, M. and Srivastava, S. Epigenetics in cancer: implications for early detection and prevention. Lancet Oncol., 3, 755–763, 2002. 9. Diaz, J.J., Giraud, S., and Greco, A. Alteration of ribosomal protein maps in herpes simplex virus type 1 infection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 771, 237–249, 2002. 10. Greco, A., Bausch, N., Coute, Y., and Diaz, J.J. Characterization by two-dimensional gel electrophoresis of host proteins whose synthesis is sustained or stimulated during the course of herpes simplex virus type 1 infection. Electrophoresis, 21, 2522–2530, 2000. 11. Greco, A., Bienvenut, W., Sanchez, J.C., et al. Identification of ribosome-associated viral and cellular basic proteins during the course of infection with herpes simplex virus type 1. Proteomics, 1, 545–549, 2001. 12. Laurent, A.M., Madjar, J.J., and Greco, A. Translational control of viral and host protein synthesis during the course of herpes simplex virus type 1 infection: evidence that initiation of translation is the limiting step. J. Gen. Virol., 79, 2765–2775, 1998. 13. Gallo, R.C. Thematic review series. XI: Viruses in the origin of human cancer. Introduction and overview. Proc. Assoc. Am. Phys,, 111, 560–562, 1999. 14. Wallace, D.C. Mitochondrial diseases in man and mouse. Science, 283, 1482–1488, 1999. 15. Enns, G.M. The contribution of mitochondria to common disorders. Mol. Genet. Metab., 80, 11–26, 2003. 16. Maechler, P. and Wollheim, C.B. Mitochondrial function in normal and diabetic beta-cells. Nature, 414, 807–812, 2001.
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- 42. 17 2 ProteomicsTechnologies and Bioinformatics Sudhir Srivastava and Mukesh Verma CONTENTS 2.1 Introduction: Proteomics in Cancer Research...............................................17 2.1.1 Two-Dimensional Gel Electrophoresis (2DE)...................................17 2.1.2 Mass Spectrometry.............................................................................18 2.1.3 Isotope-Coded Affinity Tags (ICAT) .................................................19 2.1.4 Differential 2DE (DIGE) ...................................................................19 2.1.5 Protein-Based Microarrays ................................................................20 2.2 Current Bioinformatics Approaches in Proteomics.......................................23 2.2.1 Clustering ...........................................................................................24 2.2.2 Artificial Neural Networks.................................................................25 2.2.3 Support Vector Machine (SVM)........................................................25 2.3 Protein Knowledge System............................................................................26 2.4 Market Opportunities in Computational Proteomics.....................................26 2.5 Challenges ......................................................................................................27 2.6 Conclusion......................................................................................................28 References................................................................................................................28 2.1 INTRODUCTION: PROTEOMICS IN CANCER RESEARCH Proteomics is the study of all expressed proteins. A major goal of proteomics is a complete description of the protein interaction networks underlying cell physiology. Before we discuss protein computational tools and methods, we will give a brief background of current proteomic technologies used in cancer diagnosis. For cancer diagnosis, both surface-enhanced laser desorption ionization (SELDI) and two-dimensional gel electrophoresis (2DE) approaches have been used.1,2 Recently protein-based microarrays have been developed that show great promise for analyz- ing the small amount of samples and yielding the maximum data on the cell’s microenvironment.3–5
- 43. 18 Informatics in Proteomics 2.1.1 TWO-DIMENSIONAL GEL ELECTROPHORESIS (2DE) The recent upsurge in proteomics research has been facilitated largely by stream- lining of 2DE technology and parallel developments in MS for analysis of peptides and proteins. Two-dimensional gel electrophoresis is used to separate proteins based on charge and mass and can be used to identify posttranslationally modified proteins. A major limitation of this technology in proteomics is that membrane proteins contain a considerable number of hydrophobic amino acids, causing them to precip- itate during the isoelectric focusing of standard 2DE.6 In addition, information regarding protein– protein interactions is lost during 2DE due to the denaturing conditions used in both gel dimensions. To overcome these limitations, two-dimen- sional blue-native gel electrophoresis has been used to resolve membrane proteins. In this process, membrane protein complexes are solubilized and resolved in the native forms in the first dimension. The separation in the second dimension is performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which denatures the complexes and resolves them into their separate subunits. Protein spots are digested with trypsin and analyzed by matrix-assisted laser ionization desorption time-of-flight mass spectrometry (MALDI-TOF MS). The 2DE blue-native gel electrophoresis is suitable for small biological samples and can detect posttranslational modifications (PTMs) in proteins. Common PTMs include phosphorylation, oxidation and nitrosation, fucosylation and galactosylation, reaction with lipid-derived aldehydes, and tyrosine nitration. Improvements are needed to resolve low-molecular-mass proteins, especially those with isoelectric points below pH 3 and above pH 10. This technique has low throughput (at the most 30 samples can be run simultaneously), and most of the steps are manual. Automatic spot-picking also needs improvement. 2.1.2 MASS SPECTROMETRY Mass spectrometry (MS) is an integral part of the proteomic analysis. MS instruments are made up of three primary components: the source, which produces ions for analysis; the mass analyzer, which separates the ions based on their mass-to-charge ratios (m/z); and the detector, which quantifies the ions resolved by the analyzer. Multiple subtypes of ion sources, analyzers, and detectors have been developed, and different components can be combined to create different instruments, but the principle remains the same— the spectrometers create ion mixtures from a sample and then resolve them into their component ions based on their m/z values. Significant improvements have been made in spectrometric devices during the past two decades, allowing precise analysis of biomolecules too fragile to survive earlier instrumentation. For ionization of peptides and proteins, these ionization sources are usually coupled to time-of-flight (TOF)2,7,8 spectrometers. Historically, MS has been limited to the analysis of small molecules. Larger biomolecules, such as peptides or proteins, simply do not survive the harsh ionization methods available to create the ions. ESI (electrospray ionization),9 MALDI, and SELDI techniques permit a gentler ionization of large biomolecules, called soft ionization, without too much fragmentation of the principal ions. ESI and MALDI were both developed during the late 1980s and were the foundation for the emergence of MS as a tool of investigation of biological samples. Although MALDI equipment is
- 44. Proteomics Technologies and Bioinformatics 19 expensive, quantitative high throughput can be achieved (about 100 samples per day can be run by a single laboratory). SELDI, developed in the early 1990s, is a modification of the MALDI approach to ionization. All the ionization techniques described above are sensitive in the picomole-to-femtomole range that is required for application to biological samples, carbohydrates; oligonucleotides; small polar molecules; and peptides, proteins, and posttranslationally modified proteins. Tandem mass analyzers are instruments used for detailed structural analysis of selected peptides. An example of this kind of analyzer is ABI’s QSTAR® (Applied Biosystems, Foster City, CA), a hybrid system that joins two quadrupoles in tandem with a TOF analyzer.10 Particular tryptic peptide fragments can be sequentially selected and subfragmented in the two quadrupoles, and then the subfragments can be measured in the analyzer. The resulting pattern is somewhat like the sequence-ladder pattern obtained in DNA sequencing. Although the analysis of the protein pattern is more complex than DNA sequencing, software is available that allows the direct determination of the amino acid sequence of peptides. Based on the peptide sequence information, it is possible to identify the parent protein in the database. 2.1.3 ISOTOPE-CODED AFFINITY TAGS (ICAT) Isotope-coded affinity tags (ICAT)11 is a technology that facilitates quantitative pro- teomic analysis. This approach uses isotope tagging of thiol-reactive group to label reduced cysteine residues, and a biotin affinity tag to isolate the labeled peptides. These two functional groups are joined by linkers that contain either eight hydrogen atoms (light reagent) or eight deuterium atoms (heavy reagent). Proteins in a sample (cancer) are labeled with the isotopically light version of the ICAT reagent, while proteins in another sample (control) are labeled by the isotopically heavy version of the ICAT reagent. The two samples are combined, digested to generate peptide fragments, and the cysteine-containing peptides are enriched by avidin affinity chromatography. This results in an approximately tenfold enrichment of the labeled peptides. The peptides may be further purified and analyzed by reverse-phase liquid chromatography, fol- lowed by MS. The ratio of the isotopic molecular mass peaks that differ by 8 Da provides a measure of the relative amounts of each protein in the original samples. This technology is good for detection of differentially expressed proteins between two pools. Recently the method has been modified to include 16O and 18O water and culture cells in 12C- and 13C-labeled amino acids. Problems with ICAT include its dependency on radioactive materials, its low throughput (about 30 samples per day), it only detects proteins that contain cysteine, and labeling decreases over time (see also Chapter 16). 2.1.4 DIFFERENTIAL 2DE (DIGE) Differential 2DE (DIGE) allows for a comparison of differentially expressed proteins in up to three samples. In this technology, succinimidyl esters of the cyanine dyes, Cy2, Cy3, and Cy5, are used to fluorescently label proteins in up to three different pools of proteins. After labeling, samples are mixed and run simultaneously on the same 2DE.12 Images of the gel are obtained using three different excitation/emission filters, and the ratios of different fluorescent signals are used to find protein differences among the
- 45. 20 Informatics in Proteomics samples. The problem with DIGE is that only 2% of the lysine residues in the proteins can be fluorescently modified, so that the solubility of the labeled proteins is maintained during electrophoresis. An additional problem with this technology is that the labeled proteins migrate with slightly higher mass than the bulk of the unlabeled proteins. DIGE technology is more sensitive than silver stain formulations optimized for MS. SYPRO Ruby dye staining detects 40% more protein spots than the Cy dyes. 2.1.5 PROTEIN-BASED MICROARRAYS DNA microarrays have proven to be a powerful technology for large-scale gene expression analysis. A related objective is the study of selective interactions between proteins and other biomolecules, including other proteins, lipids, antibodies, DNA, and RNA. Therefore, the development of assays that could detect protein-directed interactions in a rapid, inexpensive way using a small number of samples is highly desirable. Protein-based microarrays provide such an opportunity. Proteins are sep- arated using any separation mode, which may consist of ion exchange liquid chromatography (LC), reverse-phase LC, or carrier ampholyte–based separations, such as Rotophor. Each fraction obtained after the first dimensional separation can be further resolved by other methods to yield either purified protein or fractions containing a limited number of proteins that can directly be arrayed or spotted. A robotic arrayer is used for spotting provided the proteins remain in liquid form throughout the separation procedure. These slides are hybridized with primary anti- bodies against a set of proteins and the resulting immune complex detected. The resulting image shows only these fractions that react with a specific antibody. The use of multidimensional techniques to separate thousands of proteins enhances the utility of protein microarray technology. This approach is sensitive enough to detect specific proteins in individual fractions that have been spotted directly without further con- centration of the proteins in individual fraction. However, one of the limitations of the nitrocellulose-based array chip is the lack of control over orientation in the immobilization process and optimization of physical interactions between immobi- lized macromolecules and their corresponding ligands, which can affect sensitivity of the assay. Molecular analysis of cells in their native tissue microenvironment can provide the most desirable situation of in vivo states of the disease. However, the availability of low numbers of cells of specific populations in the tissue poses a challenge. Laser capture microdissection (LCM) helps alleviate this matter as this technology is capable of procuring specific, pure subpopulations of cells directly from the tissue. Protein profiling of cancer progression within a single patient using selected longi- tudinal study sets of highly purified normal, premalignant, and carcinoma cells provides the unique opportunity to not only ascertain altered protein profiles but also to determine at what point in the cancer progression these alterations in protein patterns occur. Preliminary results from one such study suggest complex cellular communication between epithelial and stroma cells. A majority of the proteins in this study are signal transduction proteins.5 Protein-based microarrays were used in this study. Advantages and disadvantages of some proteomic-relevant technologies are listed in Table 2.1.
- 46. Proteomics Technologies and Bioinformatics 21 TABLE 2.1 Comparisons of Various Proteomic Technologies Characteristics ELISA 2DE PAGE IsotopeCoded Affinity Tag (ICAT) TM Multidimensional Protein Identification Technology (MudPIT) TM Proteomic Pattern Diagnostics Protein Microarrays Chemiluminescence or fluorescence-based 2DE serological proteome analysis (SERPA); 2DGE + serum immunoblotting ICAT/LC-EC-MS/MS; ICAT/LCMS/MS/MALD I 2D LC-MS/MS a MALDI-TOF; SELDI-TOF; SELDIT-OF/QStar TM Antibody arrays: chemiluminescence multi-ELISA platforms; glass fluorescence based (Cy3Cy5); tissue arrays Sensitivity Highest Low, particularly for less abundant proteins; sensitivity limited by detection method; difficult to resolve hydrophobic proteins High High Medium sensitivity with diminishing yield at higher molecular weights; improved with fitting of high-resolution QStar mass spectrometer to SELDI Medium to highest (depending on detection system) Direct identification of markers N/A Yes Yes Yes No; possible with additional high-resolution MS Possible when coupled to MS technologies; or probable, if antibodies have been highly defined by epitope mapping and neutralization Use Detection of single, well- characterized specific analyte in plasma/serum, tissue; gold standard of clinical assays Identification and discovery of biomarkers not a direct means for early detection in itself Quantification of relative abundance of proteins from two different cell states Detection and ID of potential biomarkers Diagnostic pattern analysis in body fluids and tissues (LCM); potential biomarker identification Multiparametric analysis of many analytes simultaneously (Continued)
- 47. 22 Informatics in Proteomics TABLE 2.1 Comparisons of Various Proteomic Technologies (Continued) Characteristics ELISA 2DE PAGE IsotopeCoded Affinity Tag (ICAT) TM Multidimensional Protein Identification Technology (MudPIT) TM Proteomic Pattern Diagnostics Protein Microarrays Throughput Moderate Low Moderate/low Very low High High Advantages/ drawbacks Very robust, well-established use in clinical assays; requires well-characterized antibody for detection; requires extensive validation not amenable to direct discovery; calibration (standard) dependent; FDA regulated for clinical diagnostics Requires a large number of samples; all identifications require validation and testing before clinical use; reproducible and more quantitative combined with fluorescent dyes; not amenable for high throughput or automation; limited resolution, multiple proteins may be positioned at the same location on the gel Robust, sensitive, and automated; suffers from the demand for continuous on-the-fly selection of precursor ions for sequencing; coupling with MALDI promises to overcome this limitation and increase efficiency of proteomic comparison of biological cell states; still not highly quantitative and difficult to measure subpg/ml concentrations Significantly higher sensitivity than 2D- PAGE; much larger coverage of the proteome for biomarker discovery; not reliable for low abundance proteins and low-molecular-weight fractions SELDI protein identification not necessary for biomarker pattern analysis; reproducibility problematic, improved with QStar addition; revolutionary tool; 1-2 μl of material needed; upfront fractionation of protein mixtures and downstream purification methods necessary to obtain absolute protein quantification; MALDI crystallization of protein can lack reproducibility and be matrix dependent; high MW proteins requires MS/MS Format is flexible; can be used to assay for multiple analytes in a single specimen or a single analyte in a number of specimens; requires prior knowledge of analyte being measured; limited by antibody sensitivity and specificity; requires extensive crossvalidation for antibody crossreactivity; requires use of an amplified tag detection system; requires more sample to measure low abundant proteins; needs to be measured undiluted Bioinformatic needs Moderate, standardized Moderate; mostly home grown, some proprietary Moderate Moderate Moderate to extensive; home grown, not standardized Extensive, home grown; not standardized a LCM: Laser Capture Microdissection
- 48. Proteomics Technologies and Bioinformatics 23 2.2 CURRENT BIOINFORMATICS APPROACHES IN PROTEOMICS Most biological databases have been generated by the biological community, whereas most computational databases have been generated by the mathematical and computational community. As a result, biological databases are not easily acqui- escent to automated data mining methods and are unintelligible to some computers, and computational tools are nonintuitive to biologists. A list of database search tools is presented in Table 2.2, and some frequently used databases to study protein-protein interaction are shown in Table 2.3. A number of bioinformatic approaches have been discussed elsewhere in the book (see Chapters 10 and 14); therefore, we have described only the basic principles of some of these approaches. An important goal of bioinformatics is to develop robust, sensitive, and specific methodologies and tools for the simultaneous analysis of all the proteins expressed by the human genome, referred to as the human proteome, and to establish “bio- signature” profiles that discriminate between disease states. Artifacts can be intro- duced into spectra from physical, electrical, or chemical sources. Each spectrum in TABLE 2.2 Database Search Tools for 2DE and MS Name of the Software Web Site Delta2Da www.decodon.com/Solutions/Delta2D.html GD Impressionista www.genedata.com/productsgell/Gellab.html Investigator HT PC Analyzera www.genomicsolutions.com/proteomics/2dgelanal.html Phortix 2Da www.phortix.com/products/2d_products.htm Z3 2D-Gel Analysis Systema www.2dgels.com Mascot www.matrixscience.com MassSearch www.Cbrg.inf.ethz/Server/MassSearch.html MS-FIT www.Prospector.ucsf.edu Peptldent www.expasy.ch/tools/peptident.html a Software for 2DE. TABLE 2.3 Database for Protein Interaction Name of the Database Web Site CuraGen Portal.curagen.com DIP Dipdoe-mbi.ucla.edu Interact Bioinf.man.ac.uk/interactso.htm MIPS www.mips.biochem.mpg.de ProNet Pronet.doublewist.com
- 49. 24 Informatics in Proteomics MALDI or SELDI-TOF could be composed of three components: (1) true peak signal, (2) exponential baseline, and (3) white noise. Low-level processing is usually used to disentangle these components, remove systematic artifacts, and isolate the true protein signal. A key for successful biomarker discovery is the bioinformatic approach that enables thorough, yet robust, analysis of a massive database generated by modern biotechnologies, such as microarrays for genetic markers and time-of-flight mass spectrometry for proteomic spectra. Prior to a statistical analysis of marker discovery, TOF-MS data require a pre-analysis processing: this enables extraction of relevant information from the data. This can be thought of as a way to standardize and summarize the data for a subsequent statistical analysis. For example, based on some eminent properties of the data, pre-analytical processing first identifies all protein signals that are distin- guishable from noise, then calibrates mass (per charge) values of proteins for poten- tial measurement errors, and finally aggregates, as a single signal, multiple protein signals that are within the range of measurement errors. The above discussion is specifically relevant to serum-based analysis prone to all types of artifacts and errors. Serum proteomic pattern analysis is an emerging technology that is increasingly employed for the early detection of disease, the measurement of therapeutic toxicity and disease responses, and the discovery of new drug targets for therapy. Various bioinformatics algorithms have been used for protein pattern discovery, but all studies have used the SELDI ionization technique along with low-resolution TOF-MS anal- ysis. Earlier studies demonstrated proof-of-principle of biomarker development for prostate cancer using SELDI-TOF, but some of the studies relied on the isolation of actual malignant cells from pathology specimens.13–16 Body-fluid-based diagnos- tics, using lavage, effluent, or effusion material, offers a less invasive approach to biomarker discovery than biopsy or surgical-specimen-dependent approaches.17 Additionally, serum-based approaches may offer a superior repository of biomarkers because serum is easy and inexpensive to obtain.18–21 Several preprocessing and postprocessing steps are needed in the protein chip data analysis. For data analysis we must process the mass spectra in such a way that it is conducive to downstream multidimensional methods (clustering and classifica- tion, for example). The binding to protein chip spots used for general profiling is specific only to a class of proteins that share a physical or chemical property that creates an affinity for a given protein chip array surface. As a result, mass spectra can contain hundreds of protein expression levels encoded in their peaks. Bioinformatics tools have promise in aiding early cancer detection and risk assessment. Some of the useful areas in bioinformatics tools are pattern clustering, classification, array analysis, decision support, and data mining. A brief application of these approaches is described below. 2.2.1 CLUSTERING Two major approaches to clustering methods are bottom-up and top-down. An example of the bottom-up approach includes hierarchical clustering where each gene has its own profile.22 The basis of the clustering is that closest pairs are clustered
- 50. Other documents randomly have different content
- 51. those whose homes lay in foreign parts, those closely connected with the diplomatic service and the growth of the British Empire.[55] The Chronicle was a portion of the Magazine sure of finding readers, but there was no page more welcome to all than the brief but pithy preface in which the editor named the chief contents, touched on some matter of note to the readers, or urged forward the lagging subscriber. As the College interest widened with the ever-increasing number of old pupils, the Chronicle became too limited a record to stand alone. When the Magazine was about seventeen years old ‘Parerga’ appeared for the first time, telling of activities which lay outside the immediate scope of College work, yet were due in part to the influence of the Alma Mater, to ‘the spiritual force, the higher volition and action.’ Miss Beale, who found in the Magazine a strong link with her large scattered family, also in later years freely printed letters she received from various members abroad. She did not care much for articles on travel, writing on one occasion that she received too many descriptions, and would like in their place to have more records of observation in the fields of natural history and other sciences. But she treasured letters, and showed them widely. Indeed, it was sometimes startling for the writer of a private letter to Miss Beale to find whole extracts published in the Magazine for all the world to see. Almost from the beginning there were reviews of books. These were generally written by the editor. There were also notices of books by old pupils. Of these Miss Beale was proud, and she never failed to mention them, often reprinting portions of reviews by the press; but she would not review them herself, saying, ‘Books by old pupils claim our notice; we must leave criticism to those less interested in the writers.’ Fortunately Miss Beale was not content with merely reviewing and editing. Many a number of the Magazine contained a long contribution from herself, such as an article reprinted from another periodical, an address given at a gathering of old pupils, or at some
- 52. more general meeting. The first two editions of the History of the College were also printed here. Of her articles which were not of special College interest, the most notable were those upon Browning. One of these, written in spring 1890, shortly after the poet’s death, contains a brief clear statement of the value of his philosophy. The other writers of the Magazine have been chiefly old pupils, some of whose names, as, for example, those of Jane Harrison, Beatrice Harraden, Bertha Synge, May Sinclair, are known in wider fields of literature. But any who made a sincere effort were welcomed, encouraged, and—edited. Present pupils have rarely written, but of late an attempt has been made to secure more contributions from these. Members of the Council, and others connected with the College by the ties of friendship or work, frequently helped the Magazine with papers or verses. For years every number was enriched with a poem or article from the pen of Mrs. James Owen, that friend whose keen intellectual interests and strong sympathy were put so largely at Miss Beale’s service when this literary venture was first made. To find contributors Miss Beale went even beyond the outer circle of the College. ‘We always hope to have some good writing in our Magazine, thus to maintain a high standard,’ she had said at the beginning. She liked to gain the notice of those who were eminent in literature or science for this dearly loved literary child, and as occasion brought her in contact with any who were distinguished for the things she appreciated she would send them the Magazine, often asking for a paper. Letters from people of widely differing thought and position, acknowledging the receipt of the Magazine, are now in the College archives. They vary in warmth and interest. The late Bishop of Gloucester and Bristol wrote in 1889: ‘However busy I may be, I always find time to read portions of [the Magazine], and I am always thankful to recognise not merely the cultivated, but the wise and—what we men specially value—the womanly tone that characterises it. I read with much interest your article on the Sorbonne gathering.’ Bishop Westcott in 1890 wrote, on receiving the number containing Miss Beale’s ‘In Memoriam’ article on
- 53. Browning: ‘May I confess that when the copy of the Ladies’ College Magazine came this morning with the letters, my correspondence was at once interrupted? I felt constrained to read your words on Browning, just and wise and helpful and suggestive.’ Some notes are little more than the acknowledgment of a polite friend who had ‘already cut the pages.’ The request for contributions was not always granted; sometimes it was won by a little importunity. It brought about rather an amusing incident with Mr. Ruskin, whose letters on the subject and on some of Miss Beale’s own Magazine articles are too characteristic to be omitted. Miss Beale sent him the number containing her paper on ‘Britomart.’ He replied at once:—
- 54. ‘March 12, 1887. ‘Have you not yet to add to your Britomart, at p. 219, due justification of Feminine—may we not rather call it Disguise—than Lie? And, for myself, may I say that I think Britomart should have sung to the Red Knight, not he to Britomart.—Ever faithfully yours, J. Ruskin.’ Five days later he wrote:— ‘But I much more than like your essay on Britomart. ‘I am most thankful to have found the head of a Girls’ College able to do such a piece of work, and having such convictions and aspirations, and can only assure you how glad I shall be to find myself capable of aiding you in anything.... I trespass no further on you to-day, but have something to say concerning ball-play as a Britomartian exercise, before saying which, however, I will inquire of the Librarian what ground spaces the College commands, being so limited in its bookshelves.—And believe me, ever your faithful servt., John Ruskin.’ Miss Beale replied to this by sending her paper on ‘Lear,’ to which came this response:— ‘March 22, 1887. ‘I am entirely glad to hear of the Oxford plan, which seems faultless, and am most happy to get the King Lear, though I hope you have never learned as much of human life as to be able to read him as you can Britomart. What I want to know is whether Cordelia was ever so little in love—with any body, except her Father.’
- 55. Two days later came the following:— ‘March 24, 1887. ‘I have been reading your Lear with very great interest. It is one of the subtlest and truest pieces of Shakespeare criticism I ever saw, but just as I guessed—misses the key note. You never enter on the question what it is that drives Lear mad! And throughout you fall into the fault which women nearly always commit if they don’t err on the other side,—of always talking of love as if it had nothing to do with sex.... I am extremely glad to note your interest in and knowledge of music.—Ever faithfully and respectfully yours, J. Ruskin.’ After this letter there was a pause in a correspondence which had been kept up pretty briskly on various subjects. In June, however, Miss Beale wrote again,—the purport of her letter may be gathered from the answer. ‘June 8, 1887. ‘I never have been ill this year; the reports you heard or saw in papers were variously malicious or interested. But I have been busy, in very painful or sorrowful business—at Oxford or at home—nor even in the usual tenor of spring occupation could I have answered rightly the different questions you sent me. Especially, I could not tell you anything of your paper on Lear, because I think women should never write on Shakespeare, or Homer, or Æschylus, or Dante, or any of the greater powers in literature. Spenser, or Chaucer, or Molière, or any of the second and third order of classics—but not the leaders. And you really had missed much more in Lear than I should like to tell you.
- 56. ‘I really thought I had given the College my books—but if I haven’t, I won’t—not even if you set the Librarian to ask me; for it does seem to me such a shame that a girl can always give her dentist a guinea for an hour’s work, and her physician for an opinion; and she can’t give me one for what has cost me half my life to learn, and will help her till the end of hers to know. ‘Please go on with your book exactly as you like to have it. I have neither mind nor time for reading just now.— Ever most truly yrs., J. Ruskin.’ Mr. Ruskin permitted the reprint of a few extracts from his own writings in the Magazine, on which his criticism as a whole was not very encouraging. One of his letters, indeed, called forth a protest from Miss Beale, to which he replied thus:— ‘June 15, 1887. ‘Dear Miss Beale,—I am grieved very deeply to have written what I did of your dear friend’s verses. If you knew how full my own life has been of sorrow, how every day of it begins with a death-knell, you would bear with me in what I will yet venture to say to you as the head of a noble school of woman’s thought, that no personal feelings should ever be allowed to influence you in what you permit your scholars either to read or to publish.’ And again a few days later:— ‘Brantwood, Coniston, Lancashire, June 19, 1887. ‘Dear Miss Beale,—So many thanks, and again and again I ask your pardon for the pain I gave you. I had no idea of the kind of person you were, I thought you were merely clever and proud.
- 57. ‘These substituted verses are lovely.—Ever gratefully (1) yrs., ‘J. R. ‘(1) I mean, for the way you have borne with my letters. You will not think it was because I did not like my own work to have the other with it that I spoke as I did.’ Mr. Shorthouse also once contributed to the Magazine, sending a little story called ‘An Apologue.’ The work entailed by the Magazine was, on the whole, pleasant and interesting to its editor. But she was grieved sometimes if she thought old pupils did not appreciate it, or if contributions fell short. It was not always easy to get enough articles of the kind she desired, and the difficulty was increased by the severe censorship she exercised. ‘About one hour wasted in fretting over Magazine,’ runs the diary of April 2, 1891. The Magazine was not without its faults. ‘How bad the best of us!’ says Punch, according to Ruskin. But it had the conspicuous merit of offering encouragement to young writers, of promoting a spirit of unity, and fostering sympathetic interest among those whose lives were necessarily far apart. ‘We hope,’ Miss Beale had said in her first preface, ‘that the papers on work may be helpful in suggesting ways of usefulness.’[56] This hope was practically realised. How far the young writers profited by each other’s thoughts can be less easily gauged; but doubtless some learned at least one lesson the Magazine was meant to teach, that if they intended to work, they ‘must not shrink from the hardest and most fruitful work, i.e. thinking.’[57] Miss Beale’s influence was again extended in manifold and ever- developing ways when, in 1883, the first meeting of former pupils was held in the College. At this date the number of regular pupils was five hundred. Only six years before a proposal had been made to limit the numbers to three hundred, but each year saw an increase, and a consequent addition to
- 58. the ranks of those who carried the influence of the College into the larger world outside. It had been felt for some time by the Principal and others to whom the College was dear, that an association of old pupils should be formed, but of what nature and name could not be determined without a representative meeting. A suitable occasion for this presented itself in 1883, which was a sort of Jubilee year for the College, Miss Beale having then been its Principal for twenty-five years. Many old pupils expressed a wish to mark the great occasion by a personal gift to Miss Beale; she, as was to be expected, asked that it might be given to her ‘husband,’ the College. It was a moment of almost unsullied prosperity, as could be seen by the buildings which were constantly growing more stately and suitable. In the previous year they had been much enlarged, and the whole College life benefited by the addition of the Music and Art wing. The old music-rooms were little better than cupboards, the new ones contained light, air, and space, as well as the necessary pianoforte. The first drawing-room was but an insufficient classroom, in which a cast of any size could not be placed. The new studio was spacious and properly lighted. Both additions at this period spoke of Miss Beale’s method in educational development, also of the order in which her own full mental life unfolded. First she would have the exact, the severe, the discipline of grammar and rule, then the expansion of beauty in thought and symbol. And the gift of the old pupils could not have been better chosen. It took the form of an organ for what was then the largest hall, the First Division Room. Here the daily prayers of the three divisions took place. Sir Walter Parratt settled the specifications for the organ, which was placed above the Lady Principal’s dais. The choir, which up to this time had been dependent on the aid of a harmonium, was augmented and improved, and the daily music at the school prayers became a feature of College life in which Miss Beale took delight. Occasionally her directions to the choir were embarrassing. She liked music to be very piano, and required a great deal of expression to bring out the full meaning of the words sung. Mr. Ruskin was also momentarily interested by it. He was as suggestive and dogmatic on the subject as on any other that he
- 59. touched. Once he wrote to Miss Beale, ‘All music properly so called is of the Celestial Spheres. It aids and gives law to Joy, or it ennobles and comforts Sorrow.’ On hearing of the organ and ‘girl-organist,’ he hoped ‘to be able to work out some old plans with her,’ and unfolded them thus:— ‘I think you may be willing to help me in the plan chiefly for the last four or five years in my mind, of getting a girls’ choral service well organised in a college chapel. The most beautiful service I have ever heard in any church of any country is that of the Convent of the Trinità at Rome, entirely sung by the sisters, unseen; and quite my primary idea in girl education—peasant or princess, is to get the voice perfectly trained in the simplest music of noblest schools. Finding your organist is a girl, and that she is interested in the book on Plain Chant I sent her, it seems to me my time has come, and I am going to write to Miss Lefevre at Somerville, Miss Gladstone at Newnham, and Miss Welch at Girton, to beg them to consider with you what steps they could take to this end. If you could begin by giving enough time for the training of the younger girls, I think I could, with that foundation, press for a more advanced action in the matter at Cambridge and Oxford.’ Miss Beale obviously replied to this with some questions about the training of the choir, for Mr. Ruskin’s next and rapidly following letter closes thus:— ‘As for the choir, nothing is necessary but a due attention to girls’ singing, as well as their dancing. It ought to be as great a shame for a girl not to be able to sing, up to the faculty of her voice, might I say, as to speak bad grammar. You could never rival the Trinità di Monte, but could always command the chanting of the psalms with sweetness and clearness, and a graceful Te Deum and Magnificat.’
- 60. Besides the organ, Miss Beale’s wedding gifts included the first light of a stained-glass window above the new grand staircase. This was drawn by Miss Thompson, and executed by Clayton and Bell. Miss Beale herself chose the subject for the whole—a series of scenes from her beloved story of ‘Britomart.’ Over and above the opening of the new buildings, and the installation of the wedding gifts, there was in the early part of the summer term some excitement and much pleasant sense of preparation for the gathering of old pupils fixed for the 6th and 7th of July. Then, into the midst of the glad anticipation, came as with transcendent suddenness Mrs. Owen’s death on June 19. Hers was indeed ‘a spirit that went forth And left upon the mountain-tops of death A light that made them lovely.’ But for many the happiness of the coming meeting was marred, most of all for her in whose honour it had been largely arranged. Miss Beale made no change, but went through all the proceedings as they had been planned, dwelling never for a moment on her sense of bereavement and loss, but speaking calmly even in public of the life that had passed out of sight. The first meeting, on the evening of July 6, was a conversazione in the Upper or Second Division Hall. An unexpectedly large number of old pupils were present, and on the next day at the ordinary College prayers Miss Beale gave what was practically the first Guild address. Though made on an occasion of so much personal interest and gratification to herself, this address was remarkable not only for the piercing insight with which she ever penetrated below what was apparent or obvious, but also for what, for want of a better word, must be called its soberness. Touched, emotional as the speaker always was, keenly alive to the sense of union and communion with all lives that in the highest sense had come in contact with her own, happy in recognising the College to be a step by which souls might ascend out of mere material
- 61. interests, marking with joy its noble work in the progress of the ‘higher education’ of women, she chastened all excess of feeling by the calm sincerity with which she could contemplate ‘Even in the green, the faded tree.’ ‘Schools too,’ she said, ‘like the members of which they are composed, have their period of growth, manhood, and decay. Some tell us the first is over for us, and that we, too, have settled down into vigorous manhood. I am not so sure that we have quite done with growth, even in the outside body; but however that may be, I trust there is that among us, which is not even like the most substantial building, not like the outward form, liable to decay and death.’ Thus quietly she spoke, marking for all that heard her that there was no commonplace elation or poor ambition in her thoughts and feelings for her school. On this really momentous occasion for the College, when its members as a whole were summoned to catch a glimpse of all it could be of help and blessing in a far larger world than its own, the Principal spoke less of work accomplished than of growth, and ‘the silent witness of a beautiful life as a power to bless.’ She said less about the gifts with which the College had been enriched, than of some visible sacraments of Nature with which these gifts should bring them into touch. She dwelt specially on the great meanings of music. ‘In the Psalm of Life each is necessary to the perfection of that glorious music, which we shall hear and understand when the discords of earth have been resolved.’ In conclusion Miss Beale sketched the possibility of an association of old pupils, such as already existed in some boys’ schools, and was not wholly unknown among girls. ‘When I read of meetings of old Etonians, Rugbeians, Marlburians, and of works undertaken by them in common, and know how strong is the tie of affection which binds many of our old pupils to their Alma Mater, I have often wished there were some means of uniting us into an association.’ She named also the uses and aims of such an association. It is needless to say that though its members strive to bear in mind the objects their Principal and President put before them, rules, precisely to embody them, could not be framed. ‘Members should consider themselves united together to help in sustaining, especially in distant countries, as
- 62. high an intellectual and social standard as possible, first amongst those of their own class. Thus reading societies, mutual improvement societies, libraries, etc., would be helped on by them. They would bear in mind the College motto, “Let no man think or maintain that a man can search too far or be too well studied in the Book of God’s Word, or in the Book of God’s Works; but rather let men endeavour an endless progress and proficiency in both; only let men beware that they apply both to charity and not to grovelling; to use and not to ostentation.”[58] Some articles of their creed would be—(a) that influence radiates from a centre, and hence it is a duty all through life to continue one’s own education; (b) that the nearer we stand in intellectual and social position, the stronger are our ties to any, and the greater are our duties; (c) that the worst thing one can do with any talent one possesses is to bury it. Rules would have to be framed concerning admission.’ Miss Beale added that secretaries to the proposed association had already been appointed: Mrs. Ashley Smith for the general work and organisation, Miss Flora Ker as local secretary. This announcement of her appointment to what proved to be a very strenuous work was the first suggestion that Mrs. Smith received that she should even undertake it. In an article in the next Magazine Miss Beale unfolded her plan more fully, suggesting a few rules. She proposed further that the badge of the association should be a little brooch engraved with a figure of her beloved Britomart. The idea of a guild of old pupils was eagerly received, and a committee at once formed to deal with its organisation. In all these arrangements Miss Beale showed great strength of mind and self-control in being able to stand aside and let others work out the details of the scheme, even submitting her own judgment to that of the younger ones, whom she thought called upon to do the work. Yet she was in a true sense President of the Guild, guiding and directing where she would not command. Indeed, this ever-growing society which multiplied interests for her was largely her own inception, at a time when her
- 63. special work, the College, was also increasing rapidly. The power of mind which could keep the right hold on both is certainly rare. The first committee consisted of associates of the College and a few other old pupils. Meetings were held to draw up the organisation of the new society, and this was made known at large in a delightful article by Mrs. Ashley Smith in the Magazine for spring 1884. In this the writer adventured far enough into the future to be able to suggest the possibility, at no very distant date, of some corporate work, ‘such as is done by many boys’ schools,’ but in 1884 the time for this had not arrived for Cheltenham girls.
- 64. The Lower Hall, Ladies’ College Cheltenham from a photograph by Miss Bertha Synge. The second large gathering of old pupils, which took place on July 8 and 9, 1884, is always reckoned as the first meeting of the Guild, the association being on that occasion formally founded under the name of ‘The Guild of the Cheltenham Ladies’ College.’ It is interesting to note that what then seemed a large gathering really included less than eighty former pupils of the College; ten years later, at the fourth Guild meeting, there were nearly five hundred, and the number has increased ever
- 65. since. The daisy was chosen as an emblem for the Guild: its choice and its significance were explained by the President in her address on Saturday, July 9. In a second address at this time, given after the candidates for Guild membership had received their ‘Masonic sign,’ Miss Beale dwelt chiefly on the practical questions arising out of the existence of the new association. She spoke of the difficulty of decision among the many opinions which must necessarily exist in a large college; she hoped that ‘whatever decision might finally be arrived at, all would cheerfully submit to it, and if their own individual tastes were not in every case gratified, would find their satisfaction in giving up their own wishes for the sake of the majority. She herself had had to submit, she hoped cheerfully, to an adverse vote.’ The rules were then read. Of these it is sufficient to say here that they made it difficult for any one whose life was spent in a mere pleasure-seeking spirit to be a member of the Guild. The rules were accepted for two years, and two courses of study were suggested for junior members. In the year following these meetings, Mrs. Ashley Smith wrote an article for the Magazine on the reports received from various members and on the general working of the Guild, which by the end of 1885 numbered nearly two hundred members. This is now an old story, nor is there anything specially remarkable in the many details of work in Sunday-schools and coffee-clubs. Yet even at the time when the Guild, compared with its present self, looked little more than ‘seven maids with seven mops,’ the tale of individual work done shows that already much quiet persistent effort was being made by Miss Beale’s old girls. This association, founded on principles rather than rules, was indicative of its origin in a mind which habitually dwelt rather on being than doing. The small beginning, the gradual steady growth, the outcome of ideals and thoughts, were consistent with the whole of the College history. And to re-read the story of the foundation of the Guild is to remember once more how many quiet, unobtrusive, untiring workers have helped to make that history. In especial, the immense work and patience of the secretaries can perhaps never be adequately recognised: the labour of merely reading and tabulating the reports was considerable. ‘The General Secretary,’ wrote Mrs. Ashley Smith on one occasion, ‘on receiving the reports enters under more
- 66. than sixty different headings the occupations of all the Guild members. It will be easily understood that the task of reducing to order and collating a chaotic mass of miscellaneous information on all subjects, from the keeping of poultry to the study of Hebrew, from making the beds to organising institutes, is not a very simple affair, and that therefore an immense saving of time and trouble is effected when the proper form is used, and it does not become necessary to wade through a letter full of apologies and exculpatory remarks, before one can arrive at the gist of the report.’ On another occasion, after enumerating the different charitable and self-improving societies to which Guild members belonged, she said: ‘It almost gives one a headache to read this long list of occupations; and when at the end, hoping for a little breathing space, we come to an “odd minute society,” it puts the finishing touch to the bewildering sensation of restless activity, and one begins to wish for a “Sit-down- in-peace-and-calm-yourself Society.”’ The reports, a matter of obligation to the junior members of the Guild, were often looked over by the President, who would surprise the secretaries by her detailed knowledge of the home surroundings and characters of girls whom she hardly knew by sight. ‘What is so-and-so doing now?’ she would ask, and on being told, would say, ‘She ought to be doing more,’ or ‘less,’ and perhaps make some other criticism. Not less surprising was her memory of former discussions. ‘She never forgot,’ writes Mrs. Griffith, ‘what had been said. Sometimes she began again, continuing the conversation just where we left off, after a three months’ interval.’ The secretaries were also impressed by the way in which the President held herself bound by its smallest rules. Miss Helen Mugliston, who succeeded Mrs. Griffith as General Secretary in 1898, said Miss
- 67. Beale was ‘perfect to work under. Having given you the task, she gave also her absolute trust and support throughout the whole of it.’ The second meeting of the Guild was held in June 1886, lasting from a Friday evening to the following Tuesday morning. The President’s opening address dealt with work and duty. This year, for the first time, the Guild was also addressed by an outside speaker, the Dean of Gloucester. Mrs. Ashley Smith, in summing up her impressions of the gatherings of this year, rejoiced in the interest the members took in the proceedings. ‘We cannot,’ she added, ‘certainly be accused of a servile unanimity in opinions or in the expression of them; but I hope we are united in underlying principles.’ It was not until two years later that the sense of fellowship was strengthened, and the individual desires to help others directed by the resolve to organise a corporate work, a work in which not only all Guild members might help according to their opportunities, but in which also all old pupils and others connected with the College might be invited to join. This was formally proposed at the Guild meeting of 1888, and an idea as to what shape it might take was thrown out in a paper then read, which told for the first time something of what Miss Beale had done by means of the Loan Fund. To say that Miss Beale wished the corporate work to be of such a nature as to carry on that which she had long been doing for impecunious students, but feebly expresses what was really an earnest desire and hope. The claim she had upon the Guild, the importance that must attach to her lightest wish, was recognised; and yet,—yet, many felt that there were stronger reasons still why another kind of work should be chosen. Consequently no decision could be made at once, and those who had heard and discussed the paper parted after merely voting that the Guild ‘should undertake some corporate work.’ Among so many workers there were necessarily many ideas; the question was too important to be hastily decided, and it was resolved to give time for suggestions to be made and considered before anything final was done. The Committee appointed to consider these reduced them to three schemes of work, on which all members were asked to vote. These were:—
- 68. 1. A scheme for educating at College a few pupils who were worthy of education, but unable to pay the fees. 2. A scheme for taking over an elementary school in order to work it through teachers who had been trained in College. 3. The third scheme, which was carried, was submitted to the Guild in these words: ‘That the corporate fund be devoted to starting and supporting a mission in one of our large towns, the place to be decided by the votes of the Guild Members.’ It was but natural that President and members should have different ideas on such an occasion. Dorothea Beale, who had never ceased to hear and obey the call she had received as a girl to help women, and with them the race, by means of improved education, longed to see those she had taught and trained freely sharing with others the very same advantages they had received. The difficulties which beset her own youth were still fresh in her mind. The need for good teachers still existed. She had seen the work she wanted the Guild to take up in operation for years, knew that it did not pauperise, that it blessed giver and receiver, and was increasingly fruitful, like good seed in good ground. On the other hand, she had a profound suspicion of much charitable work of the day, thinking that ‘it will quickly perish because it does not aim at developing energy, inward power. To do for others what they ought to do for themselves is to degrade them in the order of creation.’[59] She could far more easily bear to see people suffering from hunger and nakedness than from loss of will power and sense of responsibility. This was partly, perhaps, because she did not know nor in the least realise the miseries and difficulties of extreme poverty. Miss Beale’s misgivings about the East End work were probably never quite set at rest. Writing to Mrs. Charles Robinson in 1899, she said: ‘I shall perhaps sleep two nights at St. Hilda’s East. I feel the whole question of Settlements most difficult. It was undertaken against my judgment, and yet the guidance all the way seems to point to its being right. Sisters and Deaconesses are much better for this work, yet there
- 69. are some whom we can enlist who will never join and could not join “Orders.”’ The Guild members who had been trained by their head not always acquiescingly to ‘do the next thing,’ but to think out questions, to plan carefully for the best if hardest, belonged to a new generation and had received another call. They saw how greatly educated women were needed to deal with charity organisation, with labour problems, with the children of the poor in schools and workhouses. Many of them were already at work for these. They felt, too, that they should take their part in helping to rouse others to study and work for the poor. On the other hand, they saw the need for cheap, good girls’ education to be one which was lessening every year. They had never felt it themselves, had had no struggle for training under pressure of adverse circumstances. Finally, they must have known that it was work which Miss Beale would not fail to carry on, meeting every necessity which was brought to her personal notice. On May 6, 1889, a general meeting of the Guild was held in London to consider further the lines on which the adopted scheme should be carried out. It was decided that the Guild Settlement should be made in London, in the parish of St. John’s, Bethnal Green, described by its vicar, the Rev. G. Bromby, who warmly welcomed the Cheltenham workers, as a ‘typical East End parish of the better sort.’ At this meeting the President introduced the subject by saying: ‘I trust we shall be able to try to win harmony out of notes not altogether concordant. Some of us come with a feeling of disappointment that the scheme we desired has been rejected;—I am one of these. I not only accept my defeat, I feel sure that you have sought guidance of that inward oracle which must ever be our supreme ruler, you have done what conscience bade, and so it is right. As regards my own scheme, I only allude to it to say, that having now to continue it single-handed, I cannot help you as much as I could wish, and I just refer to it to-day in the hope that you will remember it when I am no longer here.’
- 70. In these few words only did Miss Beale at the time announce her own disappointment and anxiety. There was much more she might have said, which she did in effect say in an early draft of her speech, which she fortunately did not destroy. Here her misgivings show themselves plainly. They were due to her foresight and judgment, yet it is likely that in some ways the untried workers, whom she feared were lightly taking upon themselves responsibilities to which they might prove unequal, really knew more than herself of the scope and details of the actual task before them. This is what Miss Beale wrote but did not say:— ‘It is no use concealing from you, for I could not, that I am greatly disappointed. But when I have said that, I have done; I accept the defeat. Others whose schemes have equally been rejected are suffering, thinking, perhaps, it is hard they have been met with so little sympathy. If they do not think well to join in this, no one will blame them, I hope, but will believe that they refuse because they ought not to give except as conscience requires, but let them give or spend in the best way they can all they would have bestowed on the Guild scheme of their heart’s choice. ‘This matter has brought before me many things which seem to show that our organisation needs some more distinct ideal. Like some “Topsy,” it could say in its infancy, “’spects I growed!” But when it undertakes to do something on its own account, then questions of power and how much power it should exercise, the questions of law and liberty which need to be faced, and which we shall, I trust, grow stronger and wiser in facing,—these have come before me with painful strength because as your President I had to face them. I was strongly opposed to the London scheme; I felt we were far too young, both in the age of the majority of our members, and also in the age of our organisation, to undertake such a great scheme. I had the strongest dislike to fashions in
- 71. philanthropy, and especially is it most undesirable to familiarise the young with lives led in the slums of heathen London. Only those whose faith has had years to grow strong seem called to such work. ‘I could not see the Head whom I could trust with its management, and such a centre of work could not be ruled by several equal Heads, or by a committee with almost no experience and but little individual responsibility. The whole thing seemed to me a mistake, and my heart sank as I thought of myself as President over our Guild, working what seemed an impossible scheme. Yet it is one of the first principles of education to let children who are not grounded properly make mistakes and so learn where they fail.’ Much happened to reconcile Miss Beale to the Settlement scheme. Miss Catherine Newman, as her sister had done ten years before in aid of poor students, volunteered to undertake the management of the work gratuitously, and to pay her own expenses. Miss Newman was an old College pupil and a member of the Guild. She was also a trained nurse, with long experience of work among the poor. Miss Newman’s offer and the appeal of her old friend, Mr. Bromby, had weight with Miss Beale. She felt less anxious about the efforts of her ‘children’ if safe-guarded by the experience of those she knew and trusted. Miss Newman could also sympathise with Miss Beale’s own disappointment and anxiety, while she was confident of her large-mindedness in this matter. This may be gathered from a letter she wrote to her in the course of the proceedings at this time:— ‘ ... It is very good of you to set aside your own wishes and to throw yourself into this scheme. I have thought many times since the corporate work was talked about, that the freedom both teachers and old pupils felt in proposing schemes of work spoke volumes for their confidence in your generosity. Several members of the Guild who felt drawn towards the mission scheme said to me, “If I thought Miss Beale would wish me to vote for
- 72. the Loan Fund because it was her scheme I would do so, but I believe that she would prefer that we should think for ourselves and vote for the scheme which most commends itself to us individually.” This confidence in your generosity and sense of justice struck me greatly; they knew you too well to fear for an instant that you might resent their taking a different line, and I felt sure from all I had ever known or seen of you that their confidence was not misplaced. Had you been able to unfold your scheme to them the result might have been very different, but of course it is too late now. If we were to renounce the idea of the Home for workers in the East- end, the elementary school would certainly take its place, and I am sure that you have realised ere now that it would be unjust both to the workers and the parish in which the Settlement is made to make it a temporary thing. Either it must be the corporate work of the Guild or it must be given up altogether,—at least so it seems to me. We could not expect enthusiasm either to work or support if it might be withdrawn at any moment. As regards your scheme, dear Miss Beale, I am truly sorry that it had not really a fair chance from the accident of its not being ripe yet for publicity. Two years hence might have been soon enough, yet I need not remind you that the “corporate work” was suggested by yourself. I am not afraid to say, however, that your scheme is sure of support and success, and this I trust while your powers are still unimpaired; but if, unfortunately, your strength should oblige you to limit your useful labour before it is fairly launched, I have every confidence that your friends and “children” would look upon it as a sacred legacy, which it would be their pride and pleasure to inherit from you.’ At the very moment that the Cheltenham Settlement was about to be opened in Bethnal Green, the ladies of Oxford were prepared to start one in the same district. For the convenience of both, an arrangement
- 73. was made by which the two sets of workers could live together for a time, under one head, Miss Newman, until the resources of each, and the work they were called upon to do, were better known. Mayfield House, close to St. John’s Church, was therefore taken and formally opened as a Ladies’ Settlement (at that time the second in London), on October 26, 1889. Four years later, as suddenly as her sister at Jersey House, Miss Newman died at her post. ‘What can one feel,’ wrote a friend to Miss Beale, ‘except that her death seems to seal the whole life with the heroism of service.’ This trouble was the first link in a chain of circumstances which led, in the course of three or four years, to the removal of the Settlement to Shoreditch, where it became an important branch of that work to which Miss Beale gave the title of St. Hilda’s.
- 74. CHAPTER XI ST. HILDA’S WORK ‘Thy kindred with the great of old.’ Tennyson, In Memoriam, lxxiv. Those who had often the advantage of hearing Miss Beale speak, either in general addresses to present or past pupils, or in the more regular course of literature lessons, soon learned that there were certain heroic names which had for her an almost romantic fascination. Among those of great women who influenced her imagination are specially to be remembered St. Hilda, St. Catherine of Siena, la Mère Angélique, Mme. Guyon. Of these the most dominant, the most inspiring was that of the great Northumbrian abbess, known to those whom she taught and ruled by the name of ‘Mother,’ not by virtue of her office, but on account of her signal piety and grace.[60] Hilda, the earnest student who ‘had been diligently instructed by learned men, who so loved order that she immediately began to reduce all things to a regular system.’ Hilda, the patron of the first English religious poet, ‘who obliged those under her to attend much to the reading of the Holy Scriptures; who taught the strict observance of justice and other virtues, particularly of peace and charity.’[61] This great Hilda and her work were to Dorothea Beale not merely romantic names, they were an ideal, an inspiration. And when the due time came, though for the sake of Miss Newman she hesitated for a moment over the alternative title of St. Margaret’s Hall, the name of St. Hilda was the one she chose to grace her own foundations. There are, possibly, members of the Ladies’ College who felt a pang of envy when the Students’ House became St. Hilda’s College. They could have borne to exchange the prim early Victorian title bestowed by the godfathers of 1856 for this more inspiring name.
- 75. There is, however, consolation in the thought that the Ladies’ College is still free to adopt the name of its second founder. St. Hilda’s Hall, as it was at first called, was formally opened on November 27, 1886; but its real building was a much longer process, even if dated only from Miss Margaret Newman’s death at the close of 1877. Miss Beale thought much and anxiously how she could best lay out the money which she and her staff and some friends had given in order that Miss Newman’s work might be carried on and enlarged. She advised with a few who cared for education and for the College. Among those who helped and counselled were Miss Soames, who subscribed largely to St. Hilda’s, and Mr. Brancker, some of whose letters on the subject remain. If there seems now to be little that is original in the suggestions and plans discussed by Miss Beale and Mr. Brancker, it is because they were to a great extent pioneers, and among the first to bring about a real system for attaining the educational objects they had at heart. In 1878 Mr. Brancker wrote:— ‘The object you advocate is a very desirable one, and one I have longed for many a time as an adjunct of the Ladies’ College—but while we were struggling upwards I could never see an opportune time to advocate my ideas on the subject. The means you suggest are very undesirable, to my mind at least, as partaking too much of the “charitable object” idea to commend themselves to me. ‘So necessary do I consider the future training of those who in their turns have to teach that for the present I should be inclined to treat every case on its own merits; as there may be many who may be anxious to get their education on such easy terms and yet have not the very least idea of imparting that knowledge to others, and in such cases the object you seek is not attained. ‘My idea, which is perhaps a crude one, would be that the capabilities of each pupil as regards teaching should be tested, and if she showed suitable powers she should be drafted into one of the boarding-houses, or if thought
- 76. better into a separate house; that the fees of the College in her case be remitted, and that the expense of her board be paid all or in part by the College. That for this she should engage to become a regular teacher; that the College should have the first claim on her services, and that she should pass all the necessary examinations appointed by the College. If in a boarding-house she might assist in keeping order and authority, not as a governess but as an elder pupil,—not as a spy but by moral power, keep her position, something like a præpostor in a public school; a great deal of evil might then be prevented by being nipped in the bud. Should she eventually wish to take a College degree she should be assisted by the College if she remained with them or under their control. My great object would be to get ladies to accept such a position, as there must be many who would come within the rules of the College as to position who would be very glad to have such a vocation in prospect, and the College ought to be in a position now, unless the funds have been unnecessarily squandered, to afford to assist such cases in the hope that in the future they would help it. ‘Such are my rough ideas on the subject, as I do not believe in the isolation of those who want a practical knowledge of human nature to enable them to become teachers worth their salt.’ In a second letter on the same subject Mr. Brancker said:— ‘I quite understand what you feel about this matter relating to the governess of the future, and it was only my fear that you might be unwittingly getting into troubled waters that induced me to write you at once about it. It is a very difficult question to solve, and one that wants a good deal more thought so that no mistake may be made. My plan is to take up the idea of a “pupil teacher” in Government Schools, and from that form
- 77. some plan for the education of those who aspire to be the teachers of the future. I should then carry out the idea I have always entertained of giving a preference to our own pupils, and working them up to our standard. I have always regretted that we missed Bessie Calrow, as she was a born teacher and would have delighted in the work. It seems to me that as you do not take these pupils until they are seventeen, you have a great chance among your own pupils, and would certainly know their own character better than any stranger; therefore, to any one who had passed through the College—could pass the necessary examination, and was willing to be such pupil teacher—I would pay the College fees and half the boarding-house expenses, or all if you like, and would give her a fair trial, and if at the end of twelve months, or longer as might be thought desirable, it was not satisfactory to all parties, let her depart and no harm would be done. This is a far better and more dignified position than being educated by charity; and the person enjoying it would lose nothing of her dignity, if it was not even added to by the position. If the plan is to do any good it must be grafted on to the College, and I for one should be very sorry to see that obliged to go to the public for any funds it requires to do good. I would make the pupils sign nothing on my plan, my hold upon them would be their association with the College. I can quite understand the difficulties raised by the boarding-houses about new pupils at that age, but with old ones that difficulty is at once removed; as, like the præpostors, they would have certain privileges, but at the same time they must submit to the discipline of the house. My plan may be, and no doubt is very crude, but these are the lines I should start from and feel my way tentatively, so as not to destroy the independence of the individual. Look where you get the best masters of public schools:—The man who succeeds is a scholar and very likely Fellow of his College; he may have been Bible-clerk, sizar, or undergraduate, and so has worked his way
- 78. upwards and obtained his position from hard work, thus adding to his dignity and power of teaching. And I should follow as much as possible in these tracks.’ Eventually the ideas expressed in these letters were carried out in the arrangement of St. Hilda’s, which became not only a home for pupils who could not afford the normal boarding fees, but also a residence for senior students who needed more liberty than they could have in the other houses. By this means the house was put on a self-supporting basis. Miss Beale could have borne with no other. The Loan Fund, up to this time, had been the means of assisting over a hundred students. Miss Beale now asked a few personal friends to support it, pointing out that such a means of help was far better than any system of scholarships, which she never ceased to dislike, and against which she continually spoke and wrote. Her chief objections to scholarships have been already noted.[62] She was moreover opposed to the principle of material giving involved in the system. She only cared, at any time, to give what would embrace and ennoble character. She thought it best that people should pay for advantages received, thought they would value them more, thought it made girls more careful and self-denying when first the management of money came into their own hands, to feel that it was not their own to do as they pleased with. A mere gift seemed to her like a dead thing compared with the money which, lent and returned and then lent to others, was thus used over and over again. Yet the want of response to appeals for the Loan Fund must have been partly due to a difference of opinion on its method rather than to want of sympathy with Miss Beale’s aims. There are many who feel an objection to saddling with a loan a young teacher starting on her work, or who recognise that an unpaid loan may help to lower the standard in money affairs, and on that account shrink from giving help in this way. There are few indeed who could lend money so successfully as Miss Beale could, because there are few who could so successfully command repayment. Of the first £500 advanced by the Loan Fund, £495 was repaid in a very few years. The pressure she would exercise for repayment sometimes led to the wrong notion that she cared for money for its own sake. She had at all times great skill in wringing the utmost use out of a sum of money to promote those ends for which she lived;
- 79. but in the ordinary commonplace sense she was indifferent to money and the things for which it is usually exchanged. Her own personal life was as bare of luxury when she was a rich woman as it was when her capital was reckoned in hundreds only. But she did care deeply for character, and anxiously avoided all forms of easy generosity which might injure those she sought to help. For several years before a turf was cut for St. Hilda’s College, Miss Beale was, as she would herself have expressed it, building it: student teachers were being trained in the College, and in 1881 one of these passed the Cambridge Examination in the Theory and Practice of Education. Gradually she gathered an increasing body of students in a separate house—a house which was as unlike as any could possibly be to the beautiful home which was shortly to be opened. She waited year after year for money with which to build without interrupting the work she had begun in assisted education, and for the reasons named made no public appeal for it. It was enough, she maintained, to state the real needs—to show the value of a work by the way it was done—and thus let it make its own appeal for support. She had a horror of plant which might be a mere empty shell, or which in its establishment might become a diversion of energy from spiritual work. She felt this especially in the matter of church building, as may be seen in the following extract from a letter: ‘What I disapproved of was the amount of begging for the Cathedral. I do not disapprove of it, but I think you know what I felt. However, the Bishop will do all he can to make it a strong spiritual centre. I can never get over the feeling of spiritual destitution at one very beautiful cathedral.’ It was also, perhaps less consciously, a principle not to take money except from those who were willing for her to carry out her own ideas. She wrote to one friend in 1888:— ‘As regards our Students’ Home, I have given up the idea of a public meeting. It seemed not right to refuse the offer at first. But I shall go on with the work, and I doubt not the money will come. There is such a great need for training teachers. If we had a meeting things might be said and money be given in a way which would pledge us, or be thought to pledge us, and now we shall be free.’
- 80. And again in 1884 to one who helped her Oxford scheme:— ‘I grieve over that Protestant spirit which forbids people to read books, to associate with people, who do not think precisely in their way. Is this done in Science? No; we put various theories before the student and show why we accept them. But we don’t ever want to impose our beliefs; so I want not to impose mine in religion, but to bring the learner to the “fountain of living water.” Any transferred opinion is without root, and cannot endure the storm. Teachers must, if they are to help, gain the sympathy they need by entering into the religious modes of seeing and feeling of many different souls. I think in a University town they would come in contact with various influences, and in a house like St. Hilda’s I should want thoughtful people who have gone through some of the experience of life,—old teachers to help the young. There is a little more of my dream, but I am quite content to wait. If it be God’s will that such a house should grow up, the way will be pointed out. I felt I could not say all this to you when we meet, and I have got to care that you should not misunderstand me.’ As the time to begin the actual erection of the house drew near she had no exultation over the fulfilment of a dream. Yet in the beginning of August 1885, surrounded by young teachers from her own and other schools drawn together for a Retreat and a brief educational conference, her mind was naturally full of that dream. Some few of her own thoughts about it she wrote down; such as the following, with their characteristic heading:— ‘Sunday, Aug. 2, 1885—on St. Hilda’s. Some thoughts at church. ‘God fulfils Himself in many ways. Lest one good custom should corrupt the world.
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